Graafian follicles explanted from proestrous rats before the preovulatory gonadotropin surge secreted predominantly 17beta-estradiol, and only small amounts of progestins and androgens, during 12 h of culture in hormone-free medium. Addition of ovine luteinizing hormone (NIH-LH-S18; 0.1-1.0 mug/ml) to the medium stimulated within 1-2 h the rate of accumulation of these steroids. However, accumulation of androstenedione and estradiol ceased after 4-6 h in the LH-treated cultures, whereas progesterone continued to accumulate and became the major secretory product at 6-12 h. Incubation of the follicles with LH for only 5 min resulted in significant stimulation of the accumulation of progesterone, androstenedione and estradiol during a subsequent 6-h culture period in hormone-free medium containing antibodies to LH; 30 min exposure to the hormone sufficed to elicit a maximal steroidogenic response and to induce ovum maturation in 95% of the follicles. Addition of actinomycin D (act D; 8 mug/ml) within the first hour after exposure of follicles to LH suppressed the LH-effect on progesterone but augmented the LH effect on estradiol accumulation; when addition of this inhibitor was delayed until 2 h, progesterone accumulation continued unabated for a further 10 h. By contrast, puromycin (80 mug/ml) inhibited progesterone accumulation when added to the medium at any time (1, 2 or 3 h) after the hormone. It is suggested that (i) a short-lived protein is essential for the stimulatory effect of LH on follicular steroidogenesis; (ii) the act D-sensitive product (mRNA?) required for the production of this protein is synthesized in adequate amount during the first 2 h of LH action, and is stable for at least 10 h; and (iii) LH may stimulate the production of an additional act D-sensitive regulatory protein that inhibits enzymes engaged in the cleavage of the 17-side-chain of progesterone, or cells equipped with these enzymes.