Calcitonin (CT) inhibits secretion of PRL when administered intravenously in rats and humans. It also inhibits PRL release from cultured rat anterior pituitary (AP) cells. Recent evidence suggests that CT-like immunoreactive peptide is synthesized and released from the AP gland. However, its physiological role in the regulation of PRL secretion has not been understood. Present studies tested the role of endogenous pituitary CT (pit-CT) in the regulation of PRL secretion in vivo by passive immunization. In the first group of experiments, ovariectomized (ovx) adult female rats were administered either preimmune or anti-salmon CT (sCT) serum, and their serum PRL levels were analyzed at various time points up to 3 h. A second group of experiments examined the effects of anti-sCT serum and dopamine on PRL release from cultured rate AP cells. In the next group of experiments, the regional distribution of pit-CT secretion was examined in different sections of the AP gland. In the last set, CT-like activity of AP extract was tested in neonatal rat kidney cells, which respond to CT with an increase in cAMP accumulation. These experiments also tested whether anti-sCT serum reduces AP extract-induced increase in cAMP accumulation. The results suggest that anti-sCT serum dramatically increased serum PRL levels (by 5-fold) of ovx rats within 30 min of administration. The serum PRL levels declined gradually after the peak. However, a significant increase in serum PRL levels was maintained by the anti-sCT serum for the duration of the experiment. The anti-serum also induced a significant increase in PRL release from cultured AP cells when added to the presence or absence of dopamine. The distribution profile of pit-CT within the AP gland suggests that the release of pit-CT immunoreactivity was significantly greater in the inner sections, and anti-sCT serum also caused greater increase in PRL release in these sections. Finally, AP extract and sCT stimulated cAMP accumulation in neonatal rat kidney cells, and anti-sCT serum significantly reduced AP extract-induced cAMP accumulation. These results demonstrate that pit-CT is an important regulator of tonic PRL secretion in female rats and can potently inhibit PRL secretion even in the presence of dopamine.