Uroguanylin is an endogenous peptide ligand for guanylyl cyclase-C, an apical membrane receptor predominantly located in the gastrointestinal epithelium. It regulates intestinal and renal fluid and electrolyte transport through the second messenger, cyclic GMP. Uroguanylin messenger RNA and the peptide are present in rat stomach, but the cellular source has not been identified. We separated gastric mucosal cells by size into seven fractions (F1-F7) and enriched endocrine cells into F1-F3 using counterflow elutriation. Uroguanylin messenger RNA and peptide were found in F1-F3 by Northern blot analysis and an RIA specific for rat uroguanylin. Uroguanylin-producing cells were identified as endocrine cells by immunocytochemical methods using antisera for uroguanylin, prouroguanylin, and chromogranin A, as well as by in situ hybridization cytochemistry. Double-staining showed that uroguanylin and histamine are colocalized in enterochromaffin-like (ECL) cells that release histamine, leading to the stimulation of gastric acid secretion from parietal cells. Uroguanylin is synthesized in ECL cells. These findings should contribute to elucidating the physiological functions of ECL cells and the cyclic GMP-mediated gastric ion transport mechanism.