We have previously reported that a synthetic peptide corresponding to amino acid residues 81-95 of the human (h) FSH-beta subunit inhibited binding of [125I]hFSH to bovine calf testis membranes and stimulated estradiol biosynthesis in primary cultures of rat Sertoli cells. We have now obtained several lines of evidence demonstrating in vivo effects of hFSH-beta-(81-95) on the mouse estrous cycle. 1) A single i.p. injection of 200 micrograms/g BW hFSH-beta-(81-95) significantly (p < 0.001) prolonged vaginal estrus in comparison to that in vehicle-injected control mice. 2) Vaginal smears taken at estrus from mice given hFSH-beta-(81-95) were characterized by the complete absence of epithelial casts, a hallmark of spontaneous ovulation in mice. 3) Mice receiving hFSH-beta-(81-95) had significantly (p < 0.001) lower serum estradiol at proestrus and serum progesterone at diestrus than vehicle-injected control mice. 4) The proestrous effects of estrogen on uterine ballooning and weight gain, clearly evident in vehicle-injected control mice, were not observed in mice treated with hFSH-beta-(81-95). A synthetic peptide corresponding to the carboxy-terminal region of hFSH-beta-(81-95), hFSH-beta-(90-95), inhibited binding of [125I]hFSH to bovine calf testis membranes, antagonized FSH-stimulated estradiol biosynthesis by primary cultures of rat Sertoli cells, and prolonged vaginal estrus in normally cycling mice. A synthetic peptide corresponding to the amino-terminal domain, hFSH-beta-(81-86), was inactive in vitro and had no effect on the mouse estrous cycle. The results of the present study provide additional evidence for in vivo effects of FSH-related synthetic peptides.