Depending on the estrous/menstrual cycle stage in females, ovarian-derived estradiol (E₂) exerts either a negative or a positive effect on the hypothalamic–pituitary axis to regulate the synthesis and secretion of pituitary gonadotropins, LH, and FSH. To study the role of estrogen receptor-α (ERα) mediating these effects, we assessed the relevant parameters in adult wild-type (WT) and ERα-null (αERKO) female mice in vivo and in primary pituitary cell cultures. The αERKO mice exhibited significantly higher plasma and pituitary LH levels relative to WT females despite possessing markedly high levels of circulating E₂. In contrast, hypothalamic GnRH content and circulating FSH levels were comparable between genotypes. Ovariectomy led to increased plasma LH in WT females but no further increase in αERKO females, while plasma FSH levels increased in both genotypes. E₂ treatment suppressed the high plasma LH and pituitary Lhb mRNA expression in ovariectomized WT females but had no effect in αERKO. In contrast, E₂ treatments only partially suppressed plasma FSH in ovariectomized WT females, but this too was lacking in αERKO females. Therefore, negative feedback on FSH is partially E₂/ERα mediated but more dependent on ovarian-derived inhibin, which was increased threefold above normal in αERKO females. Together, these data indicate that E₂-mediated negative feedback is dependent on functional ERα and acts to primarily regulate LH synthesis and secretion. Studies in primary cultures of pituitary cells from WT females revealed that E₂ did not suppress basal or GnRH-induced LH secretion but instead enhanced the latter response, indicating that the positive influence of E₂ on gonadotropin secretion may occur at the level of the pituitary. Once again this effect was lacking in αERKO gonadotropes in culture. These data indicate that the aspects of negative and positive effects of E₂ on gonadotropin secretion are ERα dependent and occur at the level of the hypothalamus and pituitary respectively.