Calcitonin, alpha and beta calcitonin gene-related peptides (CGRPs), amylin, and adrenomedullin belong to a unique group of peptide hormones important for homeostasis maintenance. We recently identified intermedin (IMD) as a novel member of the calcitonin/CGRP peptide family expressed in the pituitary, digestive tract, and other organs of vertebrates. Real-time PCR and immunohistochemical analysis of pituitaries from rats at different stages of development showed that intermedin is expressed in the intermediate lobe and select adrenocorticotrophs in the anterior lobe, suggesting that intermedin could function as a paracrine factor regulating anterior pituitary hormone secretion. In support of a paracrine role for intermedin in the pituitary, quantitative and in situ hybridization analyses showed the expression of intermedin receptor transcripts including the calcitonin receptor-like receptor and receptor activity-modifying proteins in the pituitary. Treatment with intermedin leads to a dose-dependent increase of prolactin release in cultured rat pituitary cells. In contrast, intermedin treatment has negligible effects on the release of GH, FSH, or ACTH. Likewise, in vivo treatment with intermedin leads to an elevation of plasma prolactin levels in conscious rats. Based on these functional characteristics, we hypothesized that intermedin could represent one of the intermediate lobe-derived prolactin-releasing factors important for prolactin regulation during reproduction. In support of this hypothesis, studies of intermedin expression in lactating and ovariectomized rats showed that pituitary intermedin transcripts in lactating animals increased to more than twofold over nonlactating controls whereas ovariectomy leads to a 90% reduction of intermedin expression in the pituitary. Of importance, subsequent treatment with 17-estradiol or diethylstilbestrol increased pituitary intermedin expression in ovariectomized rats. In addition, analysis of the proximate region of the intermedin gene promoter showed that the intermedin gene promoter contains consensus estrogen response element sequences, and estrogen treatments up-regulate the promoter reporter activity in transfected pituitary cells. Collectively, the present study indicates that intermedin represents a novel estrogen-dependent intermediate lobe-derived prolactin-releasing factor and could play important roles in the regulation of prolactin release during reproduction in females.