Recent studies indicate that low concentrations of acetaldehyde may function as the primary factor in alcoholic cardiomyopathy by disrupting Ca²⁺ handling or disturbing cardiac excitation-contraction coupling. By producing reactive oxygen species (ROS), acetaldehyde shifts the intracellular redox potential from a reduced state to an oxidized state. We examined whether the redox state modulates acetaldehyde-induced Ca²⁺ handling by measuring Ca²⁺ transient using a confocal imaging system and single RyR2 channel activity using the planar lipid bilayer method. Ca²⁺ transient was recorded in isolated rat ventricular myocytes with incorporated Fluo3. Intracellular reduced glutathione (GSH) level was estimated using the monochlorobimane fluorometric method. Acetaldehyde at 1 and 10 µM increased Ca²⁺ transient amplitude and its relative area in intact myocytes, but acetaldehyde at 100 µM decreased Ca²⁺ transient area significantly. Acetaldehyde showed a minor effect on Ca²⁺ transient in myocytes in which intracellular GSH content had been decreased against challenge of diethylmaleate (DEM) to a level comparable to that induced by exposure to about 50 µM acetaldehyde. Channel activity of the RyR2 with slightly reduced cytoplasmic redox potential from near resting state (-213 mV) or without redox fixation was augmented by all concentrations of acetaldehyde (1-100 µM) used here. However, acetaldehyde failed to activate the RyR2 channel, when the cytoplasmic redox potential was kept with a reduced (-230 mV) or markedly oxidized (-180 mV) state. This result was similar to effects of acetaldehyde on Ca²⁺ transient in DEM-treated myocytes, probably being in oxidized redox potential. The present results suggest that acetaldehyde acts as an RyR2 activator to disturb cardiac muscle function, and redox potential protects the heart from acetaldehyde-induced alterations in myocytes.