Exposure to methyl tertiary-butyl ether (MTBE), a commonly used octane booster in gasoline, has previously been shown to alter various muscle, kidney, and liver metabolic activities. In the present study, the metabolism of MTBE by liver microsomes from acetone- or phenobarbital-treated Sprague-Dawley rats was studied at concentrations of up to 5 mM MTBE. Equimolar amounts of tertiary-butanol, as measured by head-space gas chromatography, and formaldehyde were formed. The Vmax for the demethylation increased by 4-fold and 5.5-fold after acetone and phenobarbital treatments, respectively. The apparent Km value of 0.70 mM using control microsomes was decreased slightly after acetone treatment, but was increased by 2-fold after phenobarbital treatment. The metabolism of MTBE (1 mM) was inhibited by 35% by monoclonal antibodies against P450IIE1, the acetone/ethanol inducible form of cytochrome P450, suggesting a partial contribution by this isozyme. A single 18-h pretreatment of rats with 1 or 5 ml/kg MTBE (i.p.) resulted in a 50-fold induction of liver microsomal pentoxyresorufin dealkylase activity but no change in N-nitrosodimethylamine demethylase activity. These trends in activity agreed with immunoblot analysis which showed an elevation in P450IIB1 but no change in P450IIE1 levels.