Rabbit ovarian follicles were incubated with luteinizing hormone (LH) and various inhibitors of steroidogenesis in order to determine what enzymes were necessary for testosterone production. Incubations were carried out in minimum Eagle's medium: normal rabbit serum: 95: 5 with medium being changed every 15 min and stored at −15° C until assayed for testosterone by radioimmunoassay. Inhibitors and LH were added at different times after the start of the incubations. Of the inhibitors tested only SU 10603, an inhibitor of the 17α-hydroxylase enzyme completely prevented the testosterone response to LH while aminoglutethimide (inhibitor of 20α-hydroxycholesterol dehydrogenase) and U 30870 (inhibitor of 3β-hydroxysteroid dehydrogenase) only showed partial inhibition. These results suggest that cholesterol is an obligatory intermediate for the production of testosterone by rabbit ovarian follicles and that normal LH stimulated endocrine function can resume after treatment with inhibitors. The results are also in agreement with previous data using inhibitors of protein synthesis in the presence of LH.