Recent evidence demonstrates that ATP, costored with a number of hormones and neurotransmitters in secretory granules, is coreleased during exocytosis of these agents. Here, we explored the possibility that extracellular ATP subserves an autocrine and/or paracrine role in the regulation of prolactin (PRL) release by subjecting rat pituitary cells to various experimental manipulations aimed at evaluating putative interactions between ATP and mammotropes. Our results strongly support the view that ATP functions as a local regulator of PRL secretion. To be more specific, we observed that ATP is released in a predictable manner by physiologically relevant secretagogues that are reasonably targeted to mammotropes. Moreover, we found that ATP can act directly on pituitary cells to stimulate the release of PRL from most (if not all) mammotropes. Finally, we determined that antagonism or removal of ATP leads to a diminution of PRL export from pituitary cells cultured under basal or thyrotropin-releasing hormone-stimulated conditions. On the basis of these results, we propose that ATP acts locally to amplify and prolong the PRL secretory response elicited by a more traditional hypophysiotropic signal.