5'mRNA-variants of neuronal NOS (nNOS) are generated either by alternative promoter usage resulting in different mRNAs that encode for the same protein (nNOS), or alternative splicing encoding N-terminally truncated proteins (nNOSß/) 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 (PSD-95) in the rat gastrointestinal tract and analyzed the more distinct localization of nNOS protein variants in the duodenum by immunohistochemistry with C- and N-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; nNOSß) 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 nNOSß-mRNA was observed in pylorus, circular-muscle-layer of jejunum/ileum and caecum. Expression of nNOS and nNOSß cDNAs revealed proteins of approximately 155kDa and 135/125kDa, respectively. Immunohistochemistry showed a differential distribution of C- and N-terminal nNOS immunoreactivity in distinct layers of rat duodenum, suggesting a cell specific expression and distinct compartmentalization of nNOS proteins. The 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.