Previous studies have suggested that vasoactive intestinal polypeptide (VIP) is involved in regulation of PRL secretion within the pituitary gland. In order to determine whether VIP is synthesized in anterior pituitary tissue, we performed three experiments. In all experiments, anterior pituitaries were obtained from male rats. The tissue was then labeled by incubation in leucine-free minimum essential medium containing [3H]leucine, 14 microCi/ml. In Exp I, the labeled tissue was homogenized, centrifuged, and the supernatant was chromatographed on Sephadex G-50F. The fractions indicated a large peak of counts near the void volume and another peak coeluting with VIP. These latter fractions were pooled and subjected to reverse phase HPLC. Fractions from the HPLC indicated: a protein peak, VIP immunoreactivity, and maximum counts immunoprecipitated by anti-VIP serum at the retention time of synthetic porcine VIP. Exp II consisted of perifusion of labeled pituitary quarters over a 120-min period followed by an additional 60 min in the presence of 56 mM KCl. During this latter period of KCl depolarization, a large amount of 3H-labeled material was secreted. These fractions were then chromatographed on Sephadex G-50F, and the fractions coeluting with [125I]porcine VIP were subjected to immunoprecipitation with anti-VIP serum. In addition, all fractions from the Sephadex column were assayed for VIP, and the only activity was at the elution volume of [125I]porcine VIP. In Exp III, the pituitary labeling procedure included 3.6 X 10(-5) M cycloheximide. Subsequently, the tissue was perifused and the perifusate collected during the 60-min 56 mM KCl perifusion phase was pooled and immunoprecipitated with anti-VIP serum. No immunoprecipitable counts were obtained. These experiments indicate that anterior pituitary tissue synthesizes VIP on the basis of the HPLC profile and immunoprecipitation with specific anti-VIP antiserum. These results, in addition to other studies by our laboratory and others, suggest that intrapituitary VIP may be an important regulator of anterior pituitary hormone secretion, particularly PRL.