Thirty-day-old rats given a single subcutaneous injection of 5 IU pregnant mare serum gonadotropin (PMS) at 0900 h ovulated on the morning of day 33 (= estrus). However, the second ovulation did not occur until 9.4 days later. To determine the mechanism responsible for the delay in the second ovulation, in vivo and in vitro determinations of steroid and peptide hormones were compared between PMS-primed immature rats and adult cyclic rats. In PMS-primed rats, the corpora lutea (CL) produced progesterone for 2 days longer (until day 36) than the CL of the adult rat. Serum levels of 20 alpha-dihydroprogesterone, testosterone, and estradiol in PMS-primed rats were significantly lower than the corresponding values in adult rats. Serum LH was consistently lower in the PMS-primed rats. An increase in serum FSH occurred on days 36-37, which may be responsible for maturation of the follicles destined to ovulate at the second ovulation. On day 37, the nonluteal ovary of the PMS-primed rats also began to produce in vitro appreciable amounts of testosterone and estradiol. These findings suggest that the greater levels of prolactin and/or low levels of luteinizing hormone during estrus in PMS-primed rats may be responsible for the prolonged secretion of progesterone by the CL. This in turn inhibits follicular maturation, indirectly by lowering serum LH, which is reflected in reduced ability of the follicles in vitro to produce testosterone and estradiol until the CL regress.