Methyl t-butyl ether (MTBE), ethyl t-butyl ether (ETBE), and t-amyl methyl ether (TAME) are three alkoxyethers added to gasoline to improve combustion and thereby to reduce the level of carbon monoxide and aromatic hydrocarbons in automobile exhaust. Oxidative demethylation of MTBE and TAME and deethylation of ETBE by CYP enzymes results in the formation of tertiary alcohols and aldehydes, both potentially toxic. The metabolism of these three alkoxyethers was studied in a panel of 12 human liver microsomes. The relatively low apparent Km(1) was 0.25+/-0.17 (mean+/-SD), 0.11+/-0.08 and 0.10+/-0.07 mM and the high apparent Km(2) was 2.9+/-1.8, 5.0+/-2.7 and 1.7+/-1.0 mM for MTBE, ETBE and TAME, respectively. Kinetic data, correlation studies, chemical inhibition and metabolism by heterologously expressed human CYPs support the assertion that the major enzyme involved in MTBE, ETBE and TAME metabolisms is CYP2A6, with a minor contribution of CYP3A4 at low substrate concentration.