As a synthetic organic chemical, methyl tert-butyl ether (MTBE) is the most common fuel oxygenate. The increasing use of MTBE has raised concern over its safety. Previous studies in vivo revealed that MTBE could alter the male reproduction system. Therefore, the current experiments were designed to evaluate whether isolated mice spermatogenic cells in vitro were sensitive to exposure to MTBE at environmental levels, and to evaluate whether spermatogenic cells had undergone changes in morphologic, activity and viability parameters after exposure to MTBE. Spermatogenic cells in vitro were incubated with medium alone (control), 100 ppb, 10 ppm, 1000 ppm, 3000 ppm MTBE, respectively, for 6, 12, 18 h. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo liumbromide) assay, staining with fluorescein diacetate (FDA) and propidium iodide (PI), and flow cytometric analyses were used to assess MTBE toxicity on cells and DNA. The results showed that there were no significant differences between control and treatments of < or = 1000 ppm MTBE at the same time point. Although 3000 ppm MTBE could exert toxic effects directly on spermatogenic cells, environmental levels of MTBE did not exert toxic effects on cultured spermatogenic cells.