In previous studies we demonstrated the immunohistochemical expression of aromatase in pituitary cells. In order to determine whether pituitary aromatase is involved in the paracrine regulation of prolactin-producing pituitary cells and the physiological relevance of pituitary aromatase in the control of these cells, an in vivo and in vitro immunocytochemical and morphometric study of prolactin-positive pituitary cells was carried out on the pituitary glands of adult male rats treated with the aromatase antagonist fadrozole. Moreover, we analyzed the expression of mRNA for the enzyme in pituitary cells of male adult rats by in situ hybridization. The aromatase-mRNA was seen to be located in the cytoplasm of 41% of pituitary cells and was well correlated with the immunocytochemical staining. After in vivo treatment with fadrozole, the size (cellular and nuclear areas) of prolactin cells, as well as the percentage of prolactin-positive cells and the percentage of proliferating-prolactin cells, was significantly decreased. Moreover, fadrozole decreased serum prolactin levels. In vitro, treatment with fadrozole plus testosterone induced similar effects on prolactin-positive cells, inhibiting their cellular proliferation. Our results suggest that under physiological conditions aromatase P450 exerts a relevant control over male pituitary prolactin-cells, probably transforming testosterone to estradiol in the pituitary gland.