Ethyl tertiary-butyl ether (ETBE) is a fuel oxygenate used for the efficiency of motor vehicle fuels and their octane ratings. ETBE has been reported to induce liver adenomas in male rats in a 2-year bioassay at the highest inhalation concentration tested of 5000 ppm. To investigate the potential mutagenicity of ETBE in the liver, male Big Blue Fischer 344 rats were exposed for 28 consecutive days (6 h/day) to 0, 500, 1500, and 5000 ppm ETBE. The treated rats were sacrificed 3 days post-exposure and the frequencies of cII mutants were evaluated in the liver and bone marrow tissues. The mutant frequency (MF) of the liver in the negative control group was 36.3 × 10-6 and this value was not significantly different in ETBE-exposed animals (39.4, 37.3, and 45.9 × 10-6 in 500, 1500, and 5000 ppm groups, respectively). In the bone marrow, the mean MF in the negative control was 32.9 × 10-6 which was not different from the means of the exposed groups (33.8, 22.6, and 32.0 × 10-6 for groups exposed to 500, 1500 and 5000 ppm, respectively). These data, along with consistent negative response reported in the literature for other apical genotoxicity endpoints informs that mutagenicity is not likely the initial key event in the mode of action for ETBE-induced hepatocarcinogenesis in the rat.