Neuronal nitric oxide synthase (nNOS) generates NO in neurons, and heme-oxygenase-2 (HO-2) synthesizes carbon monoxide (CO). We have evaluated the roles of NO and CO in intestinal neurotransmission using mice with targeted deletions of nNOS or HO-2. Immunohistochemical analysis demonstrated colocalization of nNOS and HO-2 in myenteric ganglia. Nonadrenergic noncholinergic relaxation and cyclic guanosine 3',5' monophosphate elevations evoked by electrical field stimulation were diminished markedly in both nNOSDelta/Delta and HO-2(Delta)/Delta mice. In wild-type mice, NOS inhibitors and HO inhibitors partially inhibited nonadrenergic noncholinergic relaxation. In nNOSDelta/Delta animals, NOS inhibitors selectively lost their efficacy, and HO inhibitors were inactive in HO-2(Delta)/Delta animals.