Endothelin-1 (ET-1) is released in various cardiovascular disorders including congestive heart failure, and may modulate significantly the disease process by its potent action on vascular and cardiac muscle cell function and gene regulation. In adult mouse ventricular cardiomyocytes loaded with indo-1, ET-1 induced a sustained negative inotropic effect (NIE) in association with decreases in Ca(2+) transients. The ET-1-induced effects on Ca(2+) transients and cell shortening were abolished in diacylglycerol (DAG) kinase zeta-overexpressing mouse ventricular myocytes. A nonselective protein kinase C (PKC) inhibitor, GF109203X, inhibited the ET-1-induced decreases in Ca(2+) transients and cell shortening in concentration-dependent manners, whereas a selective Ca(2+)-dependent PKC inhibitor, Gö6976, did not affect the ET-1-induced effects. A phospholipase Cbeta inhibitor, U73122, and an inhibitor of phospholipase D, C(2)-ceramide, partially, but significantly, attenuated the ET-1-induced effects. Derivatives of the respective inhibitors with no specific effects, U73343 and dihydro-C(2)-ceramide, did not affect the ET-1-induced effects. Taken together, these results indicate that activation of a Ca(2+)-independent PKC isozyme by 1,2-DAG, which is generated by phospholipase Cbeta and phospholipase D activation and inactivated by phosphorylation via DAG kinase, is responsible for the ET-1-induced decreases in Ca(2+) transients and cell shortening in mouse ventricular cardiomyocytes.