We elucidated the contribution of endogenous pituitary adenylate cyclase-activating polypeptide (PACAP) to neurally evoked catecholamine secretion from the isolated perfused rat adrenal gland. Infusion of PACAP (100 nM) increased adrenal epinephrine and norepinephrine output. The PACAP-induced catecholamine output responses were inhibited by the PACAP type I receptor antagonist PACAP- (6-38) (30–3,000 nM) but were resistant to the PACAP type II receptor antagonist [Lys1,Pro2,5,Ara3,4,Tyr6]-vasoactive intestinal peptide (LPAT-VIP; 30–3,000 nM). Transmural electrical stimulation (ES; 1–10 Hz) or infusion of ACh (6–200 nM) increased adrenal epinephrine and norepinephrine output. PACAP-(6–38) (3,000 nM), but not LPAT-VIP, also inhibited the ES-induced catecholamine output responses. However, PACAP-(6–38) did not affect the ACh-induced catecholamine output responses. PACAP at low concentrations (0.3–3 nM), which had no influence on catecholamine output, enhanced the ACh-induced catecholamine output responses, but not the ES-induced catecholamine output responses. These results suggest that PACAP is released from the nerve endings to facilitate the neurally evoked catecholamine secretion through PACAP type I receptors in the rat adrenal gland.