Portuguese scientists reviewed the evidence on the elusive region, which is said to give women powerful orgasms when stimulated.
Many previous studies agreed it existed, but experts behind the new study failed to pinpoint its location, size or nature.
Writing in the journal Sexual Medicine, the team said: “Therefore, we must conclude that its existence remains to be scientifically proven.”
Researchers described the G-spot as being like Atlantis, a mythical city written about by Greek philosopher Plato.
The G-spot, thought to be an erogenous area in the vaginal wall, has been discussed in literature since the 11th century.
But despite widespread acceptance, a deluge of studies over the past few decades have failed to pinpoint whether it even exists.
Researchers have previously described the anatomical evidence of a G-spot ‘scant, insufficient and weak’.
But others have claimed such findings show ‘a lack of respect for what women say’, because a majority have reported having G-spot in a series of studies.
Medics at five hospitals in Portugal and one in Italy examined 32 studies carried out dating back to the 1980s to determine whether the G-spot existed.
The criteria for identifying the elusive region varied between the studies, but could include a more sensitive area and bulging or swelling upon stimulation.
The majority of women who participated in six questionnaire studies (62.9 per cent) claimed to have a G-spot.
And nearly three quarters (72.6 per cent) believed it was associated with having an orgasm.
Researchers said the findings ‘clearly show’ most women believe the G-spot exists, but ‘this belief may be biased by the current assumption that it does exist.
The G-spot was identified among the majority (55.4 per cent) of the 1,842 women who participated in seven clinical studies, which involved investigators manually stimulating participants or using a vibrator.
But among these seven papers, there were mixed results. It was identified among all women in two of the studies and none of the women in another two similar projects.
And even among nine medical imaging studies — which take detailed pictures of the inside of the body through ultrasounds or MRI scans — had contradictory results.
In nine separate anatomical studies, one author claimed to be able to systematically identify the G-spot, while another group did not find it at all.
And one neurophysiological study evaluated the electrical activity of the vagina through specialist tools and found that it increased in response to pressure, suggesting the mystery region did exist in some form.
Dr Pedro Vieira Baptista, a gynaecologist at Hospital Lusíadas in Porto, and his colleagues noted there were biases among the studies, such as some only including women who struggle to have an orgasm or involving an investigator manually stimulating a participant, which made it less likely for women to have a sexual response.
The studies considered that the G-spot existed, but there was no agreement on its location and size. So its existence ‘remains unproved’, the team concluded.
Researchers said: “Female sexuality, including orgasm, is much more complex than a mere formula including hormones, psychological aspects, culture, religion, anatomy, and previous experience.
“Most studies published so far about the G-spot favour its existence, but there is substantial disagreement even between these.’
“Unanswered questions remain: does it exist? If so, where is it located, what size is it, what is its histological nature, what is its role in female sexuality, is it associated with female ejaculation?”
Pressure women feel around having a G-spot and struggles to find it may lead to them feeling ‘inadequate or abnormal’, the medics said.
They added: “On the other hand, if it indeed exists, neglecting it may be equivalent to denying women the way to pleasurable experiences.
“The clitoris is still an unexplored continent, but the G-spot may just be another Atlantis.”
The researchers said more studies are needed, included on women’s opinions and ‘stimulation studies’ of women across different ages and ethnic groups.
It comes after a team of medics in Istanbul concluded last year that the evidence for the G-spot was ‘scant, insufficient and weak’.
They examined 17 middle-aged women and found no evidence of such a spot, but ‘a fairly even distribution’ of nerves instead.
And a study of 1,800 female twins by King’s College London in 2010 had previously concluded there was no evidence the G-spot exists.
Professor Tim Spector, an epidemiologist, who co-authored the research and is now best known lead author on the ZOE which records data on the prevalence of Covid and its symptoms, said at the time: “Women may argue that having a G-spot is due to diet or exercise, but in fact, it is virtually impossible to find real traits.
“This is by far the biggest study ever carried out and it shows fairly conclusively that the idea of a G-spot is subjective.”
But the findings sparked backlash from experts in France, who claimed the findings showed ‘a lack of respect for what women say’ and labelled it ‘totalitarian’.
Dr Vieira Baptista told MailOnline the existence of the G-spot has been so difficult to pin down because it “most likely because it does not exist.”
He said: “Most likely some women have more sensitivity in the anterior wall of the vagina, as it is very close to the clitoris.
“We should focus more in understanding the clitoris, before speculating about the G-spot — and other ;new’ spots that keep being added.
“Some people assumed the existence of the G-spot as a dogma — and when that happens it is not easy to contradict it.”