In an attempt to investigate the effect of ovulating hormone on the steroidogenesis of mature follicles in the course of ovulation, transitory changes of steroidogenesis in isolated rabbit follicles have been studied at several intervals after injection of an ovulatory dose of human chorionic gonadotropin (hCG). Five to ten follicles of approximately 1-2 mm in diameter were isolated from ovaries of a mature rabbit (2.5-3.0 kg) under streomicroscope, before and at the 3rd, 6th, 9th and 12th hours after intravenous injection of of 100 IU/kg of hCG. Follicles were incubated with 100 muCi of acetate-1-14C in 2 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) at 37 degrees C for 3 hours under 95% oxygen plus 5% carbon dioxide. Each incubation was terminated by quick freezing and stored forzen at -20 degrees C until eighty follicles had been collected for each time period before commencement of analysis. Incorporation of radioactive acetate into pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone., 20 alpha-dihydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, estrone and estradiol-17beta were analysed by the reverse dilution technique and identified in radiochemically pure form by recrystallization to constant specific activities. The steroidogenic activity of the follicles was evaluated by overall as well as fractionated incorporations. A peak in the overall incorporation of 14C- acetate into the ten steroids at the 3rd hour after hCG injection, followed by gradual decrease up to the 9th hour was observed. The incorporation decreased markedly to a minimum level at the 12th hour after hCG injection, which was below the level of preinjection control. Comparable quantitative fluctuations were found with the fractionated incorporation of 14C-acetate into the C21 and C18 steroids in the time sequence following hCG injection. However, the fractionated incorporation into C19 steroids reached to a maximum at the 6th hour after hCG injection. 5istribution patterns of incorporation among the individual steroids were varied at each interval of time. In the non-injected control, mature follicles synthesized predominantly estradiol-17beta, testosterone and androstenedione. Divergent steroids were formed from radioactive acetate at the 3rd hour after hCG injection. These included porgestogen, androgen and estrogen, but pregnenolone and 17hydroxyprogesterone were the two principal steroids produced. There was no essential difference in the steroidogenic patterns between the 6th and 9th hour, the major products being C21 and C19 steroids such as pregnenolone, 17hydroxyprogesterone, dehydroipiandrosterone and testosterone. The three androgens were the major steroids formed at the 12th hour after hCG injection. Thus the chages in the steroidogenic profile of the follicle was obvious in the course of ovulation. The basis of qualitative changes in follicular steroidogenesis during the process of ovulation have been discussed in connection with an accompanying effect of an ovulatory dose of hCG.