Abstract Mediators of neurogenic responses of the gastric fundus were studied in wild type and pituitary adenylate cyclase activating peptide (PACAP) knockout mice. Electrical field stimulation (EFS) to the circular muscle strips of the wild type mouse fundus induced a tri-phasic response, rapid transient contraction and relaxation, and sustained relaxation that was prolonged for an extended period after the end of EFS. The transient relaxation and contraction were completely inhibited by N G-nitro- l-arginine and atropine, respectively. The sustained relaxation was completely inhibited by a PACAP receptors antagonist, PACAP 6–38. The strips prepared from PACAP knockout mice exhibited a large contraction without rapid relaxation and unexpectedly, a sustained relaxation. However, the sustained relaxation was decreased to about a half of that observed in wild type mice. Anti-peptide histidine isoleucine (PHI) serum abolished the sustained relaxation in the knockout mice. The serum partially inhibited the sustained relaxation in wild type mice and PACAP 6–38 abolished the relaxation that remained after the antiserum-treatment. PHI relaxed the strips prepared from wild type mice. The relaxation was completely inhibited by PACAP 6–38. It was concluded that PACAP and PHI separately mediate the sustained relaxation in the mouse gastric fundus, and that nitric oxide and ACh mediate transient relaxation and contraction, respectively.