Endogenous nitric oxide (NO) has been known to regulate the salivary secretion and glandular blood flow. However, the distribution of nitric oxide synthase (NOS) responsible for NO synthesis has not been well studied in salivary glands. The present study was aimed to investigate the distribution of nitric oxide synthase isoforms (endothelial, neuronal, and inducible NOS). Immunohistochemistry, using monoclonal mouse anti-endothelial NOS, anti-neuronal NOS, and anti-inducible NOS, was performed in 3 major salivary glands (parotid, submandibular and sublingual gland) of the rat. Endothelial NOS (eNOS)-positive immunoreactivity was observed in arterial endothelium, striated duct, granular convoluted duct of the submandibular gland, intercalated duct, and mucous acinar cells of the sublingual gland. The eNOS-positive immunoreactivity was most prominent in the arterial endothelial layer and that of the striated and granular convoluted duct was well concentrated in columnar epithelial layer. In the intercalated duct and mucous acinus, eNOS-positive immunoreactivity was weakly detected. Neural NOS (nNOS)-positive immunoreactivity was observed in submandibular ganglion, autonomic postganglionic fiber, striated duct, granular convoluted duct, and intercalated duct. nNOS-positive immunoreactivity of the submandibular ganglion and autonomic postganglionic fiber was most prominent and that of the ductal system was well concentrated in the epithelial layer. eNOS-positive immunoreactivity was not detected either in excretory ducts or in serous acinar cells. Inducible NOS-positive immunoreactivity was not detected. There results reveal the presence of eNOS and nNOS in the salivary gland, which may be related with regulation of the glandular secretion and blood flow through synthesis and secretion of NO.