Background & Aims: Recently, a yeast 2-hybrid screen served to identify a new endothelial nitric oxide synthase (eNOS)-interacting protein (NOSIP), which causes redistribution of eNOS from the plasma membrane to intracellular compartments and reduces eNOS activity. Its in situ distribution is unknown and is reported here in comparison with tha of eNOS and neuronal NOS for the rat gastrointestinal tract. Methods: Immunofluorescence was performed on acetone-fixed cryosections by using a polyclonal antiserum raised against a NOSIP-glutathione S-transferase fusion protein; specificity was verified by Western blotting. Results: Cytoplasmic NOSIP immunoreactivity was observed in endothelial cells of some locations, e.g., the hepatic central vein, but it was mainly observed in the striated esophageal muscle; vascular, gastric, and intestinal smooth muscle; and in interstitial cells of Cajal. Nuclear NOSIP immunoreactivity was more widespread, including some myenteric neurons and several epithelial cell types of esophagus, stomach, pancreas, liver, and gut. This cellular distribution matched with that of its potential binding partner eNOS, as determined by immunohistochemistry and reduced nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry, and eNOS, but not neuronal NOS, could be coimmunoprecipitated with NOSIP from small intestine. Conclusions: NOSIP coimmunoprecipitates and is widely codistributed with eNOS in nonvascular cells in the gastrointestinal tract, suggesting an involvement of eNOS/NOSIP in the regulation of gastrointestinal secretion and motility.