This study was designed to determine the effects of FSH and LH on ovarian follicular development in adult hypophysectomized (HX) mice. Twelve days after HX, the animals received s.c. injections of ovine FSH (oFSH; 4 μg/day) or oFSH (4 μg/day) plus ovine LH (oLH; 2 μg/day) twice a day for 1 to 4 days. After 4 days of treatment with FSH alone, the number of preantral follicles (stages 1–3) increased significantly compared to that in HX controls and reached cyclic numbers; however, incorporation of [³H]thymidine into these preantral follicles as compared to HX controls did not increase. The number of healthy antral follicles (stages 4–5) and incorporation of [³H]thymidine into stage 5 follicles started to increase after only 1 day of treatment with FSH, and the number of atretic follicles concomitantly decreased. Treatment with both FSH and LH for 1 to 4 days increased the number of healthy follicles and restored DNA synthesis at all stages (1–5) to normal levels. Two days of replacement with FSH or FSH plus LH was required for follicles to attain preovulatory size (stage 6). FSH alone induced FSH and hCG receptors in granulosa cells, but without the induction of thecal LH/hCG receptors; FSH induced production of progesterone and androstenedione by stage 6 follicles, but not estradiol (E₂) accumulation in the incubation medium or in the serum. Combined FSH and LH induced hCG receptors in the theca and interstitium, and also restored follicular E₂ production to proestrous values. LH alone increased only the number of stage 2–3 follicles. Unexpectedly, LH alone also induced thecal hCG receptors as well as FSH receptors in granulosa cells of preantral and antral follicles. The present results demonstrate that FSH is essential for follicular growth at all stages and for prevention of atretic antral follicles. Both FSH and LH are necessary for regulation of follicular development and differentiation from the earliest preantral to preovulatory stages.