The intestinal absorption of metkephamid (MKA), an analog of natural [Met]enkephalin, was investigated by means of vascular perfusion of the rat small intestine. Most of the MKA administered to the jejunal loop was degraded in the lumen by enzymatic hydrolysis, whereas only 0.3 to 1.2% of it was absorbed into the vascular flow. This means that enzymatic degradation is a major barrier against the intestinal absorption of MKA. The absorption of MKA was divided into two steps, degradation and permeation, and is expressed as clearance from the intestine. The degradation clearance (CLd) of MKA was 60- to 200-fold higher than the permeation clearance (CLp), indicating the rapid hydrolysis of MKA before absorption. The absorbed fraction of MKA increased with increasing luminal MKA concentration, mainly due to an increase in CLp rather than a decrease in CLd. MKA was degraded not only before absorption but also during permeation across the intestinal epithelium. Three kinds of enzyme inhibitors were co-administered with MKA into the intestinal loop. Puromycin, an aminopeptidase M inhibitor, markedly enhanced MKA absorption by both decreasing CLd and increasing CLp, indicating the predominant role of this enzyme in MKA degradation. Bestatin, another aminopeptidase M inhibitor, also effectively suppressed the degradation of MKA before absorption, whereas it only slightly increased CLp. It was further found that bestatin was less effective in inhibiting MKA hydrolysis during permeation. Thiorphan, an enkephalinase inhibitor, had no effect on MKA absorption.