Enkephalin convertase (carboxypeptidase EH; EC 3.4.17.10), a carboxypeptidase B-like enzyme which processes hormone and neuropeptide precursors, has been characterized in the gastrointestinal tract, submandibular gland, and pancreas using a binding assay with [3H]guanidinoethylmercaptosuccinic acid. Binding to homogenates of the membrane and soluble fractions of stomach, small intestine, colon, and submandibular gland is saturable, with Kd values of about 2 nM. Partial purification of the membrane fractions reveals a Co+2-stimulated carboxypeptidase B activity which is not detectable in crude homogenates. In vitro autoradiography with [3H]guanidinoethylmercaptosuccinic acid localizes enkephalin convertase to the epithelial cells of the stomach, colon, and intestine, the islet cells of the pancreas, and the acinar cells of the submandibular gland. This localization contrasts to the distribution of enkephalins and other neuropeptides in the gastrointestinal tract and associated organs, suggesting that enkephalin convertase may serve functions other than neuropeptide and prohormone processing.