Vasoactive intestinal polypeptide (VIP) was measured by RIA in the hypophysial portal blood of rats after total hypophysectomy. The mean (+/- SE) VIP concentrations were 1332 +/- 171 pg/ml (range, 300-3868 pg/ml) under urethane anesthesia and 1735 +/- 707 pg/ml under pentobarbital anesthesia. The concentrations of VIP in peripheral plasma were, in most animals, less than 100 pg/ml. The secretion rate of VIP was not considerably changed during the blood collection. Immunoreactive VIP in hypophysial portal blood was identical to authentic VIP on gel chromatography. These findings suggest that VIP secreted from the hypothalamus may modulate pituitary function via the portal circulation. (Endocrinology 108: 395, 1981)