Aminopeptidase M (APM) was localized in the kidney and alimentary tract of guinea pigs and rats by indirect immunohistochemistry. APM was detected in the brush border of the epithelium of the proximal convoluted tubule of the kidney and of the small intestine, and it was localized to cells scattered throughout lymphoid tissue in the small intestine and colon. The gastric mucosa was unstained. APM was localized to numerous fibers supplying the myenteric plexus of the stomach, small intestine, and colon. The submucosal plexus was sparsely supplied by immunoreactive fibers. Occasional cell bodies were stained in the myenteric plexus. Staining was abolished by preabsorption of the primary antibody with APM. APM was characterized in membranes prepared from the muscle and mucosa of the guinea pig and rat stomach, small intestine, and colon by Western blotting. The major immunoreactive protein identified in membranes prepared from all tissues had an apparent molecular weight of 140, corresponding to the monomer of APM. In the brush border APM has a digestive function, whereas in neural tissue it may degrade and inactivate neuropeptides.