We have suggested that the cardioprotective effects of heme oxygenase-1 may be attributable to an inhibitory action of its product, carbon monoxide (CO) on cardiac L-type Ca²⁺ channels. CO-mediated Ca²⁺ channel inhibition arises from the ability of CO to promote generation of reactive oxygen species (ROS) from complex III of mitochondria (Dallas et al., 2008). Here, using mutagenesis in combination with whole-cell patch clamp recordings, we have probed the structural requirements of the α_1C subunit of the human L-type Ca²⁺ channel, stably or transiently expressed in HEK 293 cells. CO (applied using an established CO-releasing molecule, CORM-2 (Williams et al., 2004) at 30μM) exerts a strong inhibitory effect on the full-length splice variant (hHT) of the recombinant human cardiac L-type Ca²⁺ channel α_1C subunit (53.2±2.8% inhibition, n=10 cells). This effect was fully prevented by pre-treatment of cells with the reducing agent dithiothreitol (2mM). The rHT variant, lacking a cytoplasmic C-tail splice insert (Fearon et al., 2000), was insensitive to CO (n=10). Deletion mutagenesis studies demonstrated that a stretch of 34 amino acids (1785-1818) within the splice insert of hHT was essential for CO sensing. Given that CO inhibition of the channel arose via generation of ROS from mitochondria (Dallas et al., 2008), we investigated the potential involvement of each of the three cysteine residues (C1789, C1790 or C1810) in these effects of CO. Serine substitution of each of these residues fully prevented the effects of CO. Our data suggest CO regulates Ca²⁺ channel activity via redox modulation of one or more of three key cysteine residues in the C terminal tail of the channel. Whether or not these cysteines interact to regulate channel activity in the absence of CO remains to be determined.