Methylnaltrexone (MNTX), a selective mu-opioid receptor antagonist, functions as a peripherally acting receptor antagonist in tissues of the gastrointestinal tract. This report describes the metabolic fate of [(3)H]MNTX or [(14)C]MNTX bromide in mice, rats, dogs, and humans after intravenous administration. Separation and identification of plasma and urinary MNTX metabolites was achieved by high-performance liquid chromatography-radioactivity detection and liquid chromatography/mass spectrometry. The structures of the most abundant human metabolites were confirmed by chemical synthesis and NMR spectroscopic analysis. Analysis of radioactivity in plasma and urine showed that MNTX underwent two major pathways of metabolism in humans: sulfation of the phenolic group to MNTX-3-sulfate (M2) and reduction of the carbonyl group to two epimeric alcohols, methyl-6alpha-naltrexol (M4) and methyl-6beta-naltrexol (M5). Neither naltrexone nor its metabolite 6beta-naltrexol were detected in human plasma after administration of MNTX, confirming an earlier observation that N-demethylation was not a metabolic pathway of MNTX in humans. The urinary metabolite profiles in humans were consistent with plasma profiles. In mice, the circulating and urinary metabolites included M5, MNTX-3-glucuronide (M9), 2-hydroxy-3-O-methyl MNTX (M6), and its glucuronide (M10). M2, M5, M6, and M9 were observed in rats. Dogs produced only one metabolite, M9. In conclusion, MNTX was not extensively metabolized in humans. Conversion to methyl-6-naltrexol isomers (M4 and M5) and M2 were the primary pathways of metabolism in humans. MNTX was metabolized to a higher extent in mice than in rats, dogs, and humans. Glucuronidation was a major metabolic pathway in mice, rats, and dogs, but not in humans. Overall, the data suggested species differences in the metabolism of MNTX.