In true hermaphroditism diverse phenotypes and karyotypes are found; there are no distinctive laboratory features that can distinguish it from other intersex disorders, thus the diagnosis is made by the histological findings. Existence of Leydig cells is demonstrated by testosterone levels above the female range; however, presence of ovarian tissue cannot be ascertained because of the absence of a reliable functional test. Unless appropriate biopsies are performed or the whole gonad is removed, there is a risk of not diagnosing true hermaphroditism. To find a reliable test that can differentiate patients with true hermaphroditism from those with other intersex disorders, we investigated the estradiol (E2) response to human menopausal gonadotropins (hMG) in infants with genital ambiguity. These results were correlated with the histological findings. Eleven infants with genital ambiguity and four with a high scrotal testis were stimulated every 12 h with 2 IU/kg hMG. If E2 rose above 80 pg/mL (cut-off point), the test was discontinued; if after 7 days E2 remained below 80 pg/mL, the hMG dose was doubled and stimulation extended for 7 additional days. In five patients in whom true hermaphroditism was later histologically demonstrated, E2 rose above 80 pg/mL. In two of them, ovarian tissue was removed and hMG stimulation repeated; no response above our cut-off point was observed during the second test. The maximal E2 response to hMG in the remaining 10 individuals was 43 pg/mL; after laparotomy or gonadal biopsies no ovarian tissue was found. The hMG stimulation test can be considered a reliable and safe dynamic procedure for demonstrating the presence or absence of ovarian tissue in infants with genital ambiguity.