Follicles and corpora lutea were isolated from the ovaries of 25 patients at several stages of the normal menstrual cycle. To measure the steroidogenic enzyme activities, cell-free homogenates of the ovaries were incubated aerobically with 4-14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, androstenedione, and testosterone in the presence of appropriate cofactor(s). The delta 5-3 beta-hydroxysteroid dehydrogenase plus delta 4-delta 5 isomerase activity in the follicle was low during early follicular phase (greater than 11 days before ovulation), but gradually increased toward ovulation and showed maximal values in the corpus luteum at the midluteal phase (6-10 days after ovulation). The activities of 17 alpha-hydroxylase and C-17-C-20 lyase in the follicle reached the highest value at the late follicular phase (within 5 days of ovulation), but markedly decreased after luteinization. 17 alpha-Hydroxylase activity in the corpus luteum increased again during the midluteal phase, whereas the activity of C-17-C-20 lyase remained at a low level throughout the luteal phase. The aromatase activity was low during the follicular phase, but increased markedly with luteinization. These findings are discussed in light of the known plasma steroid concentrations and their changes during the menstrual cycle.