Estrogens increase prolactin (PRL) synthesis and release in rats and humans, whereas pituitary-derived calcitonin-like immunoreactive peptide (pit-CT) inhibits PRL gene expression and release. To test the hypothesis that estrogens stimulate lactotrophs by diminishing pit-CT expression, the present studies examined effects of ovariectomy (ovx) and estradiol (E2) administration on (1) pit-CT IR cell population; (2) pit-CT IR content and (3) release of pit-CT IR by cultured anterior pituitary (AP) cells. Ability of anti-calcitonin immunoglobulins (anti-CT IgG) to stimulate PRL release from cultured AP cells was also examined. The results suggest that ovariectomy induced a large increase in pit-CT IR cell population in the AP gland and E2-treatment dramatically reversed this increase. Similar changes were observed in pit-CT IR content of AP extracts. Cultured AP cells from ovx rats released significantly higher amounts of pit-CT IR, and anti-CT IgG induced a significant increase in basal PRL release. AP cells from E2-treated rats secreted lower amounts of pit-CT IR and this was associated with significantly higher PRL release. These results suggest that estrogens may stimulate lactotrophs, at least in part, by removing inhibitory influence of endogenous pit-CT.