Our previous study has demonstrated that instead of neuromuscular blockage cartap, an organonitrogen insecticide, could cause a marked irreversible Ca 2+-dependent contracture in both isolated mouse and rabbit phrenic nerve-diaphragms. We further examined the potential of direct myocytotoxicity of cartap and the possible roles of calcium ion and oxidative stress on cartap-induced muscle cell injury using the mouse myoblast cell line, C 2C 12. Cartap exerted a dose- and time-dependent cytotoxic effect in C 2C 12 cells measured by MTT colorimetric assay and trypan blue dye exclusion. The extracellular activities of both creatine kinase (CK) and lactate dehydrogenase (LDH) were elevated in the cartap-treated groups at or greater than 100 μM. The isoenzymatic profiles showed that the elevations were mainly due to CK-3, LDH-3, and LDH-4. Following the addition of 0.5–2.5 mM EGTA, a Ca 2+ chelator, or 30–100 μM verapamil, an L-type Ca 2+ channel blocker, the cartap-induced reduction in MTT metabolic rate of C 2C 12 cells was significantly restored in a dose-dependent manner in both EGTA and verapamil-treated cells. Furthermore, EGTA could significantly reduce the cartap-induced elevation in the levels of total extracellular CK and LDH activities. Additionally, cartap significantly increased the level of endogenous reactive oxygen species (ROS) in C 2C 12 cells in a dose- and time-dependent manner. The cartap-induced ROS generation could be significantly inhibited by antioxidants, including Vitamins C and E, catalase, and superoxide dismutase, with catalase the most effective. EGTA could significantly inhibit cartap-induced ROS generation in a dose-dependent manner. The results suggested that cartap could induce ROS generation in C 2C 12 cells via a Ca 2+-dependent mechanism resulting in subsequent cytotoxicity, at least partially, to C 2C 12 cells. It is speculated that both Ca 2+ and Ca 2+-induced ROS may also play the central role on the myogenic contracture and myofiber injury of the diaphragm leading to respiratory failure and subsequent death in rabbits exposed ocularly to cartap.