Vasoactive intestinal peptide (VIP)-immunoreactive nerve fibers have been demonstrated in the rat adrenal cortex in close association with zona glomerulosa cells, suggesting neural regulation of adrenocortical cell function (5). The present studies were undertaken to study the possible role of VIP in the regulation of steroid hormone secretion from the outer zones of the normal rat adrenal cortex. Capsule-glomerulosa preparations, consisting of the capsule, zona glomerulosa, and a small but variable portion of the zona fasciculata, were perifused in vitro. To assess the secretory responsiveness of the capsule-glomerulosa preparation, aldosterone and corticosterone release were measured after stimulation with ACTH and angiotensin II. ACTH (10(-12)-10(-8) M) stimulated dose-dependent increases in aldosterone secretion (1.9- to 36.9-fold increases over basal values) and corticosterone secretion (1.4- to 14.0-fold increases over basal values). Angiotensin II (10(-8)-10(-5) M) stimulated dose-dependent increases in aldosterone secretion (1.6- to 8.8-fold increases over basal values). VIP (10(-6)-10(-4) M) stimulated dose-dependent increases in both aldosterone (1.7- to 41.0-fold) and corticosterone secretion (1.8- to 5.3-fold). However, glucagon and (N-Ac-Tyr1-D-Phe2)GRF-(1-29)NH2, peptides structurally related to VIP, stimulated neither aldosterone nor corticosterone secretion, indicating that VIP effects are likely to be specific for this peptide. It is noteworthy that in this preparation, the stimulation of corticosteroid secretion by VIP at 10(-5) and 10(-4) M was comparable to those by 10(-6) M angiotensin II and 10(-9) M ACTH, respectively. These results support the hypothesis that the VIP innervation of the adrenal cortex may contribute directly to the regulation of adrenal steroidogenesis.