Creatine is a major component of energy metabolism and enzymes involved in its synthesis have therefore been of considerable interest. L-arginine-glycine amidinotransferase, commonly called transamidinase, catalyzes the first reaction in the biosynthesis of creatine. This first reaction is believed to occur in the kidney because of the high concentration of transamidinase in that tissue. Transamidinase activity is also found in many other tissues of the rat, but its role in these tissues is not known. Immunochemical studies with antisera and monoclonal antibodies were used to confirm and refine our understanding of the presence of transamidinase in rat tissues. Immunofluorescence histochemistry was performed to localize transamidinase immunoreactivity within specific tissues including cells in the proximal tubules of the kidney, hepatocytes of the liver, and alpha cells of the pancreatic islet. Immunochemical studies with monoclonal antibodies confirm localization of transamidinase immunoreactivity in the proximal tubules of the kidney. The localization of such immunoreactivity in specialized cells yields insight into possible physiological role(s) of transamidinase in the rat.