Steroid hormones regulate levels of gonadotropin mRNA in the pituitary, and gonadotropic hormones in plasma. To determine whether estrogen receptor alpha (ERalpha) mediates steroid negative feedback, wild type (WT) and estrogen receptor alpha knockout (ERalphaKO) mice of both sexes were gonadectomized and implanted with a Silastic capsule containing either estradiol (E2), dihydrotestosterone (DHT), testosterone, or a blank capsule. Ten days later, plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were measured. Pituitary mRNA levels of gonadotropin subunit (alpha, LHbeta, FSHbeta) and prolactin (PRL) were quantified. LH levels in gonad-intact ERalphaKO females were elevated, similar to values seen following gonadectomy. By contrast, serum LH concentrations in gonad-intact ERalphaKO males were low and rose following gonadectomy, suggesting androgen feedback. Estradiol treatment significantly decreased plasma LH in WT animals, but not in ERalphaKOs. In fact, in female ERalphaKOs, our dose of E2 increased plasma levels of LH as compared with untreated, ovariectomized ERalphaKOs. All the steroid treatments suppressed LH in WT animals whereas only DHT consistently suppressed LH concentrations in ERalphaKO mice. The postgonadectomy rise in plasma FSH was prevented by steroid treatments in WT females, but not in any of the other groups. Gonadotropin subunit and PRL mRNA responses to E2 treatment (both inhibitory and stimulatory) were absent in ERalphaKO mice, suggesting a critical role for ERalpha. Although E2 can exert negative feedback effects on LH release in both males and females by actions at the ERalpha, the androgen receptor plays the primary physiological role in the male mouse.