Alcohol dehydrogenase (ADH), which oxidizes ethanol into acetaldehyde, exacerbates ethanolinduced cardiac depression although the mechanism of action remains unclear. This study was designed to examine the impact of antioxidant catalase (CAT) on cardiac contractile response to ethanol and activation of stress signaling. ADH-CAT double transgenic mice were generated by crossing CAT and ADH lines. Mechanical, intracellular Ca²⁺ properties and reactive oxygen species (ROS) generation were measured in ventricular myocytes. ADH-CAT, ADH, CAT and wild-type FVB myocytes exhibited similar mechanical and intracellular Ca²⁺ properties. ADH or ADH-CAT myocytes had higher acetaldehyde producing ability. Ethanol (80-640 mg/dl) suppressed FVB cell shortening and intracellular Ca²⁺ transients with maximal inhibitions of 43.5% and 45.2%, respectively. Ethanol-induced depression on cell shortening and intracellular Ca²⁺ was augmented in ADH group with maximal inhibitions of 66.8% and 69.6%, respectively. Interestingly, myocytes from CAT-ADH mice displayed normal ethanol response with maximal inhibitions of 46.0% and 47.2% for cell shortening and intracellular Ca²⁺, respectively. CAT transgene lessened ethanol-induced inhibition on cell shortening (maximal inhibition of 30.3%) but not intracellular Ca²⁺. ADH amplified ethanol-induced ROS generation which was nullified by the CAT transgene. Western blot analysis showed that ethanol reduced ERK phosphorylation and enhanced JNK phosphorylation without affecting p38 phosphorylation. The ethanol-induced changes in phosphorylation of ERK and JNK were amplified by ADH. CAT transgene itself did not affect ethanol-induced response in ERK and JNK phosphorylation but it cancelled ADHinduced effects. These data suggest that antioxidant CAT may effectively antagonize ADHinduced enhanced cardiac depression in response to ethanol.