In cardiac myocytes, hypoxia inhibits the basal L-type Ca ²⁺ current ( I _Ca-L ) and increases the sensitivity of I _Ca-L to β-adrenergic receptor stimulation. We investigated whether hydrogen peroxide (H ₂ O ₂ ) is involved in the hypoxic response. Guinea pig ventricular myocytes were dialyzed with catalase, which specifically catalyzes the conversion of H ₂ O ₂ to H ₂ O and oxygen, and then I _Ca-L was recorded during exposure to isoproterenol (Iso). Catalase decreased the K _0.5 for activation of I _Ca-L by Iso from 2.7±0.3 nmol/L (in cells dialyzed with heat-inactivated catalase) to 0.4±0.1 nmol/L. The increase in sensitivity to Iso by catalase may be attenuated when cells are preexposed to H ₂ O ₂ . A significant increase in sensitivity of I _Ca-L to Iso was recorded when mitochondrial function was inhibited with myxothiazol or FCCP, suggesting that the source of H ₂ O ₂ was from the mitochondria. Prior exposure of cells to H ₂ O ₂ attenuated the inhibition of basal I _Ca-L during hypoxia and the increase in sensitivity of I _Ca-L to Iso during hypoxia. Additionally, extracellularly applied catalase mimicked the effect of hypoxia on basal I _Ca-L . Measurement of the rate of production of hydrogen peroxide using 5- (and 6-)chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate acetyl ester indicated that hypoxia was associated with a significant decrease in the production of hydrogen peroxide in the cells. These data suggest that hypoxia mediates changes in channel activity through a lowering in H ₂ O ₂ levels and that H ₂ O ₂ is a key intermediate in modifying basal channel activity and the β-adrenergic responsiveness of the channel during hypoxia.