Moderate alcohol consumption has been shown to reduce the morbidity and mortality from coronary heart disease. Ethanol elicits its protective effects via mechanisms that include activation of protein kinases linked to growth and survival. Our results in isolated neonatal rat cardiomyocytes demonstrate that repeated short-term, low-dose exposure to ethanol is sufficient to activate the growth and/or survival pathways that involve PKC-epsilon, Akt, and AMP-activated kinase. In addition, we are able to induce apoptosis in these cardiomyocytes using the saturated fatty acid palmitate. Pretreatment with multiple low-dose ethanol exposures attenuates the apoptotic response to palmitate. This protection is manifested by a reduction in caspase-3-like activity, decreased mitochondrial loss of cytochrome c, and decreased loss of the mitochondrial lipid cardiolipin. We previously reported that incubation of cardiomyocytes with palmitate results in decreased production of reactive oxygen species compared with cells incubated with the nonapoptotic fatty acid oleate. In the present study, we observed an increase in the production of superoxide and the rates of fatty acid oxidation in cardiomyocytes pretreated with ethanol and then exposed to fatty acids. The level of superoxide production in palmitate-treated cells returns to the levels observed in oleate-treated cells after ethanol exposure. Taken together with our observed increase in AMP-activated kinase activity, we propose that ethanol pretreatments stimulate oxidative metabolism and electron transport within cardiomyocytes. We postulate that stimulation of palmitate metabolism may protect cardiomyocytes by preventing accumulation of unsaturated precursor molecules of cardiolipin synthesis. Maintaining cardiolipin levels may be sufficient to prevent the mitochondrial loss of cytochrome c and the downstream activation of caspases.