The physiological importance of gonadal hormones in feedback control of gonadotropin secretion during the estrous cycle in golden hamsters was investigated with immunoneutralization methods. Anti-inhibin serum (inhibin-AS) treatment always induced a drastic increase in follicle-stimulating hormone (FSH) secretion and occasionally raised luteinizing hormone (LH) secretion. Anti-estradiol-17 serum (estradiol-AS) treatment increased LH secretion typically. Although estradiol-AS elevated FSH secretion occasionally, the elevation was much less than that by inhibin-AS. Plasma FSH reached ovariectomized levels by a synergistic effect of both antisera. Elevated plasma LH with both antisera was much less pronounced than in ovariectomized animals. Plasma LH increased dramatically to the levels in the ovariectomized group when antibody against progesterone (progesterone-AB) was given together with inhibin-AS and estradiol-AS, although progesterone-monoclonal antibody alone did not alter plasma gonadotropin levels. These results indicate that in hamsters FSH secretion is mainly regulated by inhibin and LH secretion is regulated by estradiol-17 and progesterone during the estrous cycle.