Estrogens play an important role in the growth, differentiation, and function of female reproductive tissues. Estrogen signals through estrogen receptors (ERs), members of the nuclear receptor superfamily. The two major forms, ERalpha and ERbeta, are expressed in the mouse ovary, where ERbeta is predominantly expressed in granulosa cells, and ERalpha in theca cells. In this study, we determined the expression pattern of ER subtypes within mouse follicles cultured from the early preantral stage up to the preovulatory stage and after an ovulatory stimulus in different culture conditions. Immunohistochemical studies performed at different time points of culture revealed that ERbeta was found exclusively in granulosa cell nuclei regardless of follicular growth stage or culture conditions. In contrast, ERalpha was found in oocyte, granulose, and theca cells, and its subcellular localization differed between follicular growth stages and culture conditions. A shift from a predominant cytoplasmic to a predominant nuclear immunolocalization was observed in granulosa cells as follicles reached the antral growth phase, and was postponed in culture conditions with minimal growth factor supplementation. In response to hCG, ERbeta protein levels in luteinized granulosa cells spectacularly declined to undetectable levels, while ERalpha immunostaining again shifted to cytoplasmic regions, but not in theca cells.