To determine the mechanisms responsible for the suppression of follicular development in the lactating rat, in vivo and in vitro determinations of steroid and peptide hormones combined with the histological size-distribution of ovarian follicles were compared among three groups of post-parturient rats nursing 0, 2 or 8 pups. From Days 4 to 12 (day of parturition = Day 0 of lactation), in dams nursing 8 pups, follicles larger than 401 microns in diameter were absent, whereas follicles of this size were always present in mothers nursing 0 or 2 pups. The in vitro production rates of estradiol-17 beta (E2) and testosterone (T) by nonluteal ovarian tissues (NLO) were consistently lower in dams nursing 8 pups than in mothers nursing 0 or 2 young, correlating with the histological observations. Serum E2 and 20 alpha-dihydroprogesterone (20 alpha-OHP) in rats with 8 pups were significantly lower, whereas serum progesterone (P) was significantly increased compared to the values in dams nursing 0 or 2 pups. In mothers nursing 8 pups, the corpora lutea (CL) of lactation, formed at postpartum ovulation, produced in vitro significantly greater amounts of P but significantly lesser amount of E2 compared to the other groups. Serum luteinizing hormone (LH) was consistently lower in dams nursing 8 pups than in dams nursing 0 or 2 pups. There was, however, no significant difference in serum follicle-stimulating hormone (FSH) among the 3 types of postparturient rats. Throughout lactation serum values of FSH were within the range of diestrous levels during the estrous cycle. Serum levels of prolactin (Prl) in dams nursing 8 pups were strikingly higher compared to dams nursing 2 pups. The administration of small doses of human chorionic gonadotropin (hCG) or ovine LH twice daily from Days 2 to 5 to lactating rats nursing 8 pups induced the maturation of follicles capable of ovulating. These findings indicate that during the first half of lactation the strong suckling stimulus reduces serum LH directly or indirectly via high levels of serum Prl and P, which in turn inhibit follicular maturation. During the second half of lactation, the suckling stimulus wanes, Prl and P decline and serum LH recovers to basal cyclic values which are reflected in the initiation of follicular maturation.