Methyl tertiary butyl ether (MTBE), the main component of oxygenated gasoline, is added to gasoline as an octane enhancer to reduce automotive emissions. We evaluated the mutagenicity of domestic MTBE using the Ames test, unscheduled DNA synthesis (UDS) assay, and NIH/3T3 cell micronucleus test. MTBE did not show any mutagenic potential in the Ames assay (TA98, TA100 strains) with or without rat liver-derived metabolic activation system (S-9). In rat primary hepatocyte UDS assay, a dose-response relationship was observed, which meant that MTBE might damage normal DNA and induce unscheduled DNA synthesis. MTBE did not show positive results in the NIH/3T3 cell micronucleus test. It could be concluded that MTBE has some degree of mutagenicity.