Estrogens play a central role in regulating female reproduction throughout the reproductive axis, and the pituitary is one of the major targets of estrogen action. We hypothesized that estrogen receptor alpha (ERalpha) mediates estrogen action in the pituitary gonadotroph. To test this hypothesis, we generated a mouse line with a selective ERalpha deletion in the gonadotropin alpha-subunit (alphaGSU)-expressing pituitary cells (pituitary-specific ERalpha knockout; ERalpha(flox/flox) alphaGSU(cre)). Although the ERalpha(flox/flox) alphaGSU(cre) female mice maintain a basal level of serum LH and FSH and their ovulatory capacity is comparable to that in controls, they do not display regular estrous cycles and are infertile, indicating a potential disorder in regulating LH and/or FSH secretion. The ERalpha(flox/flox) alphaGSU(cre) female mice express equivalent levels of LHbeta and alphaGSU mRNA compared with wild-type mice as determined by microarray analysis. Taken together, these findings indicate that pituitary gonadotroph ERalpha carries out the effects of estrogens with regard to estrous cyclicity and ultimately fertility.