Uroguanylin, a member of the guanylin peptide family, acts on guanylyl cyclase C (GC-C) to regulate intestinal and renal fluid and electrolyte transport through the second messenger, cGMP. Using an antiserum raised against synthetic rat uroguanylin, we established an RIA and identified three endogenous molecular forms in the intestine and kidney: a 15-amino acid uroguanylin, an 18-amino acid uroguanylin that is a monobasic processing product, and a 9.4-kDa prouroguanylin. Prouroguanylin is the major molecular form in these two tissues, whereas only 15-amino-acid uroguanylin is present in the urine. Rat uroguanylin is most abundant in the proximal small intestine, its content decreasing toward the colon. Uroguanylin is present immunohistochemically in the endocrine cells in the intestine and stomach, B cells in the pancreatic islets, and tubular epithelial cells in the kidney. Uroguanylin has a widespread tissue distribution and is located in cells that function in an endocrine, paracrine, and/or luminocrine (luminal secretion) fashion. Uroguanylin may have physiological functions other than the regulation of fluid and electrolyte transport in the intestine and kidney.