Estrogens play a major role in the regulation of prolactin (PRL) secretion through activation of pituitary and hypothalamic estrogen receptors (ERs). In order to evaluate the relative role of ERα and ERβ in the control of PRL density in the pituitary gland, we performed immunocytochemical localization of PRL and ERs in pituitaries of wild-type (WT), ERα knockout (KO) and ERβKO mice. In WT and ERβKO anterior pituitaries, the vast majority of secretory cells contained ERα immunoreactivity, while no ERα immunostaining could be found in ERαKO pituitaries. No ERβ immunoreactivity could be detected in pituitaries of WT, ERαKO or ERβKO mice. At the light microscopic level, a large number of cells staining for PRL were present in pituitaries of female WT, while in female ERαKO pituitaries, the density of PRL cells was much lower. In WT male pituitaries, the density of PRL cells was lower than observed in female WT, while PRL staining was markedly decreased in male ERαKO as compared to male WT. In ERβKO mice of both sexes, the results were identical to those observed in WT animals. At the electron microscopic level, in WT mice of both sexes, type 1 PRL cells exhibited a well-developed Golgi apparatus and a large number of strongly stained large mature and immature secretory granules. Type 2 PRL cells were also present in the pituitary. Type 2 PRL cells contain small poorly labelled granules. In ERαKO mice of both sexes, type 1 PRL cells were atrophied with poorly developed Golgi apparatus, and no type 2 PRL cells could be observed. In ERαKO pituitaries, typical gonadectomy cells were found. No ultrastructural changes were observed in PRL cells of ERβKO mice. The present data strongly suggest that the positive regulation of PRL expression at the pituitary level by estrogens is mediated by ERα and does not involve ERβ activation.