Cultured Sertoli cells were tested for their cytotoxicity and oxidative stress induced by Methyl tert-butylether (MTBE) which has been extensively used as a gasoline additive. In cytotoxic experiments, Sertolicells were cultured with medium alone (control), 5, 500, or 50,000_M MTBE. Lactate dehydrogcnase(LDH) leakage assay, staining with fluorescein diacetate (FDA) and propidium iodide (PI), and flow cytometricanalyseswere used. In oxidative stress experiments, Sertoli cellswere cultured withmedium alone(control), 0.5, 50, or 5000_M MTBE. The production of reactive oxygen species (ROS), maleic dialdehyde(MDA) content and the level of superoxide dismutase (SOD) activity in cell supernatants were measured.Meanwhile, the expression level of 8-oxoguanine DNA glycosidase (OGG1) and extracellular form of superoxidedismutase (SODEX) in Sertoli cells were determined by RT-PCR.We also compared the current findingswith the previous findings in rat spermatogenic cells exposed to MTBE. The present data indicate thathigh dose MTBE may exert a direct toxic effect on Sertoli cells. Oxidative stress induced by MTBE is apossible mechanism of cytotoxicity.