We investigated the role of inducible nitric oxide (NO) synthase (iNOS) on ischemic myocardial damage in rats exposed to daily low nontoxic levels of carbon monoxide (CO). CO is a ubiquitous environmental pollutant that impacts on mortality and morbidity from cardiovascular diseases. We have previously shown that CO exposure aggravates myocardial ischemia-reperfusion (I/R) injury partly because of increased oxidative stress. Nevertheless, cellular mechanisms underlying cardiac CO toxicity remain hypothetical. Wistar rats were exposed to simulated urban CO pollution for 4 wk. First, the effects of CO exposure on NO production and NO synthase (NOS) expression were evaluated. Myocardial I/R was performed on isolated perfused hearts in the presence or absence of S-methyl-isothiourea (1 μM), a NOS inhibitor highly specific for iNOS. Finally, Ca 2+ handling was evaluated in isolated myocytes before and after an anoxia-reoxygenation performed with or without S-methyl-isothiourea or N-acetylcystein (20 μM), a nonspecific antioxidant. Our main results revealed that 1) CO exposure altered the pattern of NOS expression, which is characterized by increased neuronal NOS and iNOS expression; 2) cardiac NO production increased in CO rats because of its overexpression of iNOS; and 3) the use of a specific inhibitor of iNOS reduced myocardial hypersensitivity to I/R (infarct size, 29 vs. 51% of risk zone) in CO rat hearts. These last results are explained by the deleterious effects of NO and reactive oxygen species overproduction by iNOS on diastolic Ca 2+ overload and myofilaments Ca 2+ sensitivity. In conclusion, this study highlights the involvement of iNOS overexpression in the pathogenesis of simulated urban CO air pollution exposure.