Mice lacking neuronal nitric oxide synthase gene (ncNOS) were used to determine the enzymatic source of nitric oxide (NO) and its relationship with other putative inhibitory neurotransmitters. Inhibitory junction potentials (IJP) of circular smooth muscle of gastric fundus were studied. The IJP in the wild-type mice consists of overlapping components, the fast and slow IJPs. NOS inhibitor L-NA or VIP receptor antagonist VIP(10-28), blocks the slow IJP but not the fast IJP. The fast UP is blocked by alpha-beta methylene ATP tachyphylaxis, by reactive blue 2, and by apamin. The IJP in the ncNOS-deficient [ncNOS(-)] mutant is of short duration and is abolished by blockers of the fast IJP, but is unaffected by blockers of the slow UP. Exogenous VIP produces membrane hyperpolarization in strips from wild-type but not ncNOS(-) mice. The hyperpolarizing action of VIP is resistant to nifedipine but is sensitive to omega-conotoxin GVIA. In conclusion: (a) NO derived from ncNOS is an inhibitory neurotransmitter rather than a postjunctional mediator; (b) VIP is a prejunctional neurotransmitter that causes release of evanescent NO; and (c) ATP acts in parallel with the VIP/NO pathway.