The involvement of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) in nonadrenergic noncholinergic (NANC) nerve-induced relaxation and the interaction between NO and VIP were investigated in the mouse gastric fundus. N-omega-nitro-L-arginine (L-NOARG; 100 muM) completely inhibited the NANC relaxations induced by electrical stimulation (ES) (0.5, 1, 2, 4, and 8 Hz; 25 V; 1 ms; 15-s trains). Hemoglobin (20 muM), hydroxocobalamin (100 muM), and 1H-[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 muM) diminished ES-induced relaxations, but alpha -chymotrypsin (10 U/ml) and VIP antiserum (1/200 dilution) had no effect on NANC relaxations. L-NOARG (100 muM) did not have any effect, whereas ODQ (10 muM) attenuated sodium nitroprusside (SNP; 100 nM)-induced relaxations. alpha -Chymotrypsin (10 U/ml) had no effect on the response to SNIP. Furthermore, alpha -chymotrypsin (10 U/ml) abolished and VIP antiserum (1/200 dilution) diminished VIP (50 nM)-induced relaxations. L-NOARG (100 muM) caused an inhibition of VIP-induced relaxation that was reversed by L-arginine (1 mM) but not by D-arginine (1 mM). Similarly, ODQ (10 muM) inhibited the responses to VIP. 2-Amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (5 muM) had no effect on these relaxations. L-NOARG (100 muM) and ODQ (10 muM) did not affect isoproterenol (10 nM)-induced relaxations. In conclusion, these results provide evidence that NO is involved in NANC nerve-induced relaxation and the participation of VIP (and related neuropeptides) cannot be excluded in causing relaxation of mouse gastric fundus muscle strips. These findings support the idea that VIP directly stimulates the production of NO by increasing NOS activity and thereby activating soluble guanylyl cyclase in smooth muscle.