As a volatile synthetic organic chemical, methyl tert-butyl ether (MTBE) was the most common gasolineadditive. The increasing use of MTBE raised concern over its health safety. Inhalation was the principleroute of exposure for the general population. This study used a model of rabbit tracheal epithelial cells(RTEs) in primary culture to investigate the cytotoxic effects induced by MTBE and the potential mechanism.RTEs were incubated with medium alone (control), 0.5, 50, 5000 ppm MTBE respectively. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo liumbromide) assay, staining with fluorescein diacetate,propidium iodide and lactate dehydrogenase leakage ratio were used to assess MTBE cytotoxicity on cells.We also observed a significant elevation in cytosolic Ca2+ by fluorescence probe Fluo-3AM at 3, 6 and 12 hfollowing exposure to MTBE. Loss of mitochondrial membrane potential (MMP) was detected following12 and 24 h treatment of NP and assessment by rhodamine 123 (Rh123) staining. Activity changes ofthe Ca2+–ATPase, Ca2+-Mg2+-ATPase following MTBE treatment displayed a similar trend, suggestingan initial elevation before 6h and subsequent dramatic decrease at 12h. Our results demonstrated thatinduction of cell injury, associated with mitochondrial dysfunction, and alterations in cytosolic Ca2+ inRTEs represent key mechanisms by which MTBE exerts its cytotoxic effects.