The absorption characteristics of morphine were investigated by using rat gastrointestine. Absorption and transport experiments were carried out by the in situ loop and the in vitro everted sac methods, respectively. Brush border membrane vesicles (BBMVs) were used for uptake experiments. Morphine and its metabolites, morphine-3-glucuronide (M-3-G), and morphine-6-glucuronide (M-6-G), in biological samples were simultaneously determined by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection and electrochemical detection. In the in situ loop method, morphine was well absorbed in the order of jejunal site greater than duodenal site greater than ileal site greater than middle intestinal site greater than rectal site, but it was poorly absorbed from the stomach. In each of the everted duodenal and jejunal sacs, 2,4-dinitrophenol, a metabolic inhibitor, inhibited the transport of morphine from the mucosal side to the serosal side. Further, HgCl2 pretreatment reduced the absorption of morphine from the duodenal and the jejunal loops. The initial uptake of morphine by BBMVs was stimulated in the presence of an H+ gradient (inner pH 7.5 and outer pH 5.0) and an overshoot phenomenon was observed. The initial uptake showed concentration dependence, i.e., it was saturable. Results obtained in this study indicate that carrier-mediated transport stimulated by the H+ gradient is partly involved in the duodeno-jejunal absorption of morphine, although morphine is passively absorbed from other sites.