Although protein kinase C (PKC) plays a pivotal role in ischemic preconditioning, it is not clear what the end effector is that protects the myocardium. In isolated, paced (1.25 Hz, 36-37°C) adult rat cardiomyocytes, the effects of PKC preactivation by diacylglycerol on cell motion, intracellular Ca²⁺ concentration ([Ca²⁺]_i; indo 1), and intracellular pH (pH_i; seminaphthorhodafluor-1) during simulated ischemia-reperfusion (I/R) were investigated. The degree of reperfusion-induced contracture was significantly attenuated in the myocytes pretreated with 10 µM 1,2-dioctanoyl-sn-glycerol (DOG; n = 19) compared with the untreated myocytes (n = 23, P < 0.02). There were no differences in twitch amplitude, end-diastolic [Ca²⁺]_i, or peak-systolic [Ca²⁺]_i during I/R between the DOG-pretreated and untreated myocytes. Although there were no differences in pH_i during ischemia, the pH_i overshoot during reperfusion was significantly delayed in the DOG-pretreated myocytes compared with the untreated myocytes (n = 17 for each, P < 0.01). Chelerythrine completely abolished the favorable effects of DOG on the reperfusion-induced contracture and the pH_i overshoot. These data suggest that diacylglycerol attenuates I/R injury in isolated, paced cardiomyocytes, which may be related to the slower pH_i overshoot during reperfusion.