Conditions of stress such as myocardial infarction stimulate up-regulation of heme oxygenase-1 (HO-1) to provide cardioprotection, but its mechanism of action is unknown (Clarke et al., 2003). One of the products of HO-1 catabolism is carbon monoxide (CO). We have investigated the ability of CO to act as a modulator of L-type Ca²⁺ channels, using whole-cell patch clamp recordings from HEK 293 cells stably expressing the human α_1C Ca²⁺ channel subunit as previously described (Scragg et al., 2005). CO, applied via the CO donor molecule CORM-2 (1-70μM; Williams et al., 2004), caused reversible, voltage-independent Ca²⁺ channel inhibition of up to ca. 40%, whereas its inactive form (iCORM) was without significant effect. CO-mediated inhibition was independent of protein kinase G activation since effects were unaltered in cells pretreated with PET-cGMPS (100nM). Two distinct NO donors, SIN-1 (10μM) and GSNO (2mM) failed to mimic the inhibitory actions of CO and did not significantly alter Ca²⁺ currents. The actions of CO were prevented by the antioxidant MnTMPyP (100μM). Inhibition of NADPH oxidase (apocyanin; 30μM, or diphenyleneiodonium; 3μM), or xanthine oxidase (allopurinol, 1μM) did not affect the inhibitory actions of CO. Instead, inhibitors of complex III (but not complex I) of the mitochondrial electron transport chain, namely antimycin A (3μM) and stigmatellin (1μM) and a mitochondrially-targeted antioxidant (Mito Q; 250nM), fully prevented the effects of CO. Our data indicate that the cardioprotective effects of HO-1 activity may be attributable to an inhibitory action of CO on cardiac L-type Ca²⁺ channels. Inhibition arises from the ability of CO to promote generation of reactive oxygen species from complex III of mitochondria.