It is well known that the suckling stimulus renders mammotropes considerably more responsive to prolactin (PRL)-releasing stimuli, and the neurointermediate lobe peptide -melanocyte-stimulating hormone (-MSH) has been proposed to play a pivotal role in this priming. The objectives of the present study were to determine whether -MSH could act directly on pituitary cells to potentiate PRL release in response to two physiologically relevant PRL secretagogues, thyrotropin-releasing hormone (TRH) and ATP, and, if so, to identify the mechanism by which this priming phenomenon is manifested. To this end, we cultured anterior pituitary cells from lactating rats overnight and then subjected them to a reverse hemolytic plaque assay for PRL to evaluate their responses to various test agents. We found that -MSH, which had no effect on PRL export when tested alone, augmented by more than threefold the secretory responses to TRH and ATP. Next, we utilized digital-imaging fluorescence microscopy of fura 2 to evaluate the role of intracellular Ca²⁺ in this process. We found that PRL export induced by pharmacological activation of L-type voltage-operated calcium channels was also potentiated by -MSH, as was Ca²⁺ entry induced by TRH. Our results indicate that -MSH acts as a mammotrope-priming agent on a subset of mammotropes by increasing Ca²⁺ entry induced by PRL secretagogues.