Adam Ostrzenski, of the Institute of Gynecology in St Petersburg, Florida, said he has located the "anatomic existence" of the pleasure zone which is just half the size of a fingernail.
Writing in the Journal of Sexual Medicine, Ostrzenski, said it was a "well-delineated sac structure" on the perineal membrane, precisely 8.1mm long and 3.6mm wide.
"This study confirmed the anatomic existence of the G-spot, which may lead to a better understanding and improvement of female sexual function," he was quoted as saying by the Daily Telegraph.
The G-spot was named after Ernst Grafenberg, a German gynaecologist who claimed to have discovered it in 1950.
The theory has been maintained ever since by sex therapists, who have claimed some women need coaching to find it and experience the full pleasure of sex.
Despite Ostrzenski's certainty, his finding is not likely to be the final word on the matter.
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Earlier this year a rival medic, Amichai Kilchevsky of the Yale-New Haven Hospital in Connecticut, said after reviewing 100 studies that there was no conclusive evidence of its existence. He hoped it would take the pressure off men and women during sex.
Prof Kevan Wylie, a sexual medicine consultant in Sheffield and associate editor of the Journal of Sexual Medicine, said its existence could not be confirmed by a single post mortem.
There were other theories that did not rely on the existence of the G-spot to explain female orgasms, he said.
Others are disdainful of the continued fascination with female sexuality in general, and the G-spot in particular.
Petra Boynton, a lecturer in health services research at University College London, said: "We persistently try and make out there are unique parts of the female genitals that should or should not be stimulated to encourage orgasm."