Mediators of neurogenic responses of the gastric antrum were studied in wild-type and pituitary adenylate cyclase-activating polypeptide (PACAP) -knockout (KO) mice. Electrical field stimulation (EFS) to the circular muscle strips of the wild-type mouse antrum induced a triphasic response; rapid transient relaxation and contraction, and sustained relaxation that was prolonged for an extended period after the end of EFS. The transient relaxation and contraction were completely inhibited by L-nitroarginine and atropine, respectively. The sustained relaxation was significantly inhibited by a PACAP receptor antagonist, PACAP(6-38). The antral strips prepared from PACAP-KO mice unexpectedly exhibited a tri-phasic response. However, the sustained relaxation was decreased to about one-half of that observed in wild-type mice. PACAP(6-38) inhibited EFS-induced sustained relaxation (33.5% of control) in PACAP-KO mice. Anti-peptide histidine isoleucine (PHI) serum partially (the 30% inhibition) or significantly (the 60% inhibition) inhibited the sustained relaxations in the wild-type and PACAP-KO mice, respectively. The immunoreactivities to the anti-PACAP and anti-PHI serums were found in myenteric ganglia of the mouse antrum. These results suggest that nitric oxide and acetylcholine mediate the transient relaxation and contraction, respectively, and that PACAP and PHI separately mediate the sustained relaxation in the antrum of the mouse stomach.