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The concentrations of testosterone, progesterone and 20-hydroxypregn-4-en-3-one (20-OHP) were measured in the ovaries of immature rats in which ovulation was induced by treatment with pregnant mare serum gonadotrophin (PMSG) and, 48 h later, with human chorionic gonadotrophin (HCG). The concentration of testosterone in the tissue increased significantly 48 h after treatment with PMSG, reached a peak 4 h after the administration of HCG and declined to the basal level 4 h later. Increases in the levels of progesterone and 20-OHP were observed 4 h after the administration of HCG. Whereas the level of 20-OHP continued to rise during the subsequent 30 h, progesterone levels declined near the presumed time of ovulation (12 h after administration of HCG). It is concluded that 20-hydroxysteroid dehydrogenase activity is present in the immature rat ovary before ovulation and that an increase in the production of testosterone in the ovaries of rats treated with PMSG and HCG precedes increased production of progesterone and 20-OHP in these ovaries.