The toxicity of the chiral fluorinated volatile anesthetics halothane, enflurane, and isoflurane is closely related to their metabolism by hepatic cytochrome P450. Although individual anesthetic enantiomers have been shown to exhibit a difference in anesthetic efficacy, metabolism of anesthetic enantiomers has not been reported. Human liver enflurane metabolism to difluoromethoxydifluoroacetic acid (DFMDFA) and inorganic fluoride is catalyzed in vivo and in vitro by cytochrome P450 2E1. The purpose of this investigation was to characterize enflurane racemate and enantiomer metabolism to test the hypothesis that fluorinated ether anesthetic metabolism by cytochrome P450 2E1 exhibits substrate stereoselectivity. Enflurane metabolism by microsomes from three human livers and by microsomes containing cDNA-expressed human P450 2E1 was measured at saturating enflurane concentrations. DFMDFA was quantitated with gas chromatography-mass spectrometry by detection of the ethanolamide derivative. In microsomes from all three livers, (R)-enflurane metabolism was significantly greater than that of (S)-enflurane, whereas rates of racemic enflurane metabolism were generally between those seen for the R- and S-enantiomers. The ratio of (R)-enflurane to (S)-enflurane metabolism in the three livers studied was 2.1:1, 1.9:1, and 1.4:1. (R)-, (S)-, and racemic enflurane were all metabolized by expressed P450 2E1. The ratio of (R)-enflurane to (S)-enflurane metabolism was 1.9:1. The metabolic enantiomeric selectivity of human liver P450 2E1 for (R)-enflurane suggests that enflurane metabolism by P450 2E1 occurs by direct substrate oxidation, rather than indirectly through the generation of a P450-dependent reactive oxygen species, and supports the hypothesis that the P450 2E1 active site is somewhat restrictive and capable of stereochemical discrimination.