To understand the pathophysiological role of endothelin-1 in the failing heart, we constructed a cellular mitochondrial impairment model and demonstrated the effect of endothelin-1. Primary cultured cardiomyocytes from neonatal rats were pretreated with rotenone, a mitochondrial complex I inhibitor, and the cytotoxic effect of endothelin-1 on the cardiomyocytes was demonstrated. Rotenone gradually decreased the pH of the culture medium with incubation time and caused slight cell injury. Endothelin-1 markedly enhanced the effect of rotenone that decreased the pH of the medium and enhanced cellular injury. The enhancement of the decrease in pH and cell injury induced by endothelin-1 was counteracted by the endothelin ET_A receptor antagonist BQ123 or by maintaining the pH of the medium by the addition of 50 mM HEPES. Endothelin-1 markedly increased the uptake of 2-deoxyglucose and lactic acid production when the cardiomyocytes were pretreated with rotenone. These findings suggest that the stimulation of glucose uptake and anaerobic glycolysis followed by the increase in lactic acid accumulation in cardiomyocytes under the condition of mitochondrial impairment may be involved, at least in part, in the cellular injury by endothelin-1. Moreover, these findings suggest the possibility that the effect of endothelin-1 on myocardium is reversed by the condition of the mitochondria, and endogenous endothelin-1 may deteriorate cardiac failure with mitochondrial dysfunction. This may contribute to clarify the beneficial effect of endothelin receptor blockade in improving heart failures.