Methyl-tertiary-butyl ether (MTBE) is a ubiquitous oxygen-bearing additive used to reduce engine knocking and obtain cleaner gasoline combustion. Conflicting data have been reported about a possible carcinogenic role of MTBE in humans. In this study we evaluated the effects of MTBE on cell growth and transformation in rodent fibroblasts. We found that MTBE inhibits cell proliferation in a dose- and time-dependent pattern with an IC50 of about 0.84 mM. We also studied the effects of MTBE on cell cycle distribution. The most striking effect was a reduction in the percentage of cells in the G2/M-phase which was associated with an increase of cells in the S-phase of the cell cycle, as assessed by flow cytometry. At a dose corresponding to IC50, a subdiploid peak indicative of apoptosis, was also evident. MTBE was also able to induce cell transformation in vitro. In conclusion, our results suggest that MTBE can affect cell growth and induce cell transformation in cultured rodent fibroblasts.