Naltrexone was incubated with the 9000 X g supernatant of guinea pig liver in the presence of an NADPH-generating system to determine the relative amounts of the 6-keto reduction products, alpha- and beta-naltrexol, formed in vitro. After a 0.5-2 hour period both alpha- and beta-naltrexol were formed as detected by electron capture gas chromatography of pentafluoropropionic anhydride-derivatized extracts of the incubation mixture. The identity of alpha-naltrexol was confirmed by nuclear magnetic resonance, infrared and mass spectra, as well as by mixed melting point determination. The percentage of natrexol found as the alpha-epimer ranged from 36-81% and was concentration-dependent at substrate concentrations ranging from 0.0007-0.1 mM. The hepatic enzyme(s) responsible for the reduction of naltrexone were localized in the 105,000 X g supernatant fraction of guinea pig liver. In contrast to results obtained for the reduction of naltrexone using guinea pig liver, the 9000 X g and 105,000 X g supernatant fractions of monkey liver appeared to reduce naltrexone almost exclusively to beta-naltrexol. The 9000 X g supernatant of rat liver was less active than similar preparations from guinea pig or monkey liver; only a small amount of beta-naltrexol and no detectable alpha-naltrexol was formed after two hours of incubation. Incubation of the naltrexone metabolites, alpha- or beta-naltrexol, with the 9000 X g supernatant of guinea pig liver and incubation of alpha-naltrexol with guinea pig kidney slices yielded no evidence of interconversion of these metabolites.