5' mRNA variants of neuronal nitric oxide synthase (nNOS) are generated either by alternative promoter usage resulting in different mRNAs that encode for the same protein (nNOS) or alternative splicing encoding NH₂-terminally truncated proteins (nNOS/) that lack the PDZ/GLGF domain for protein-protein interaction of nNOS. We studied the expression of 5' nNOS mRNA forms and nNOS-interacting proteins (postsynaptic density protein-95; PSD-95) in the rat gastrointestinal tract and analyzed the more distinct localization of nNOS protein variants in the duodenum by immunohistochemistry with COOH- and NH₂-terminal nNOS antibodies. 5' nNOS mRNA variants showed a site-specific expression along the gastrointestinal tract with presence of all forms (nNOS-a, -b, -c; nNOS) in the muscle layer of esophagus, stomach, duodenum, longitudinal muscle layer of jejunum/ileum, proximal colon, and rectum. In contrast, a lack of nNOS-a and nNOS mRNA was observed in pylorus, circular muscle layer of jejunum/ileum, and cecum. Expression of nNOS and nNOS cDNAs revealed proteins of ~155 kDa and 135/125 kDa, respectively. Immunohistochemistry showed a differential distribution of COOH- and NH₂-terminal nNOS immunoreactivity in distinct layers of rat duodenum, suggesting a cell-specific expression and distinct compartmentalization of nNOS proteins. Observed distribution of 5' nNOS mRNA variants and proteins argue for a complex control of nNOS expression by usage of separate promoters, cell- and site-specific splicing mechanisms, and translational initiation. These mechanisms could be involved in gastrointestinal motor diseases and may explain the phenotype of nNOS knockout mice with gastric stasis and pyloric stenosis, due to a total loss of nNOS in the pyloric sphincter region.