The case of a true hermaphrodite, with a normal ovary and an ovotestis is presented. The ovotestis was removed and incubated in vitro with tritiated steroids (testosterone, dehydroepiandrosterone, pregnenolone and 17α-hydroxyprogesterone). Labeled metabolites were isolated and identified. Based upon these findings, a pathway of steroid biogenesis in this abnormal gonadal tissue is suggested. The ovotestis studied did not contain all the enzymes involved in ovarian steroidogenesis: 3 β-hydroxysteroid dehydrogenase, isomerase, 17–20 desmolase and 17 β-hydroxysteroid dehydrogenase were present, but other important enzymes, such as 16 and 17-hydroxylases, and aromatizing enzyme systems, were deficient or absent. The biosynthesis of steroids has been studied in some abnormal conditions such as testicular feminization, Klinefelter’s syndrome, polycystic ovary and cancer. In the present study, the steroid metabolism in gonadal tissue from a true hermaphrodite was investigated in vitro.