Enkephalinase (EC 3.4.24.11, membrane metalloendopeptidase) is a zinc peptidase expressed by neurons and a variety of epithelial cells, and responsible for the inactivation of enkephalins in brain. Its functions in the gastrointestinal (GI) tract are less well understood although enkephalinase inhibitors were reported to induce a constellation of antisecretory and motor responses. Its localisation in various segments of the rat GI tract was established autoradiographically using a 125I-labelled monoclonal antibody. All along the GI tract, the highest immunoreactivity was found in mucosal layers e.g., in intestinal villi, basal epithelial layers of the oesophagus or gastric cardia, muscularis mucosae of the stomach and large intestine. The immunoreactivity was also high in the stomach submucosae and moderate in the muscularis propria of the caecum. A faint patchy immunoreactivity was also observed in several other layers. This distribution suggests that the membrane peptidase is expressed by enterocytes and a variety of other cells. Its high expression in mucosal layers is consistent with its participation in protein digestion and also in the inactivation of endogenous peptides, particularly the enkephalins, acting at this level to control secretory mechanisms and hydroelectrolytic fluxes. Its presence in submucosal layers may account for some naloxone-reversible motor responses elicited by enkephalinase inhibitors.