—Elevated levels of oxygen free radicals have been implicated in the pathways of reperfusion injury to myocardial tissue. The targets for free radicals may include specific as well as random intracellular components, and part of the cellular response is the induction of extracellularly activated and stress-activated kinases. The intermediate signals that initiate these stress responses are not known. Here we show that one of the earliest responses of cardiac myocytes to hypoxia and reoxygenation is the activation of neutral sphingomyelinase and accumulation of ceramide. Ceramide increased abruptly after reoxygenation, peaking at 10 minutes with 225±40% of the control level. Neutral sphingomyelinase activity was induced with similar kinetics, and both activities remained elevated for several hours. c-Jun N-terminal kinase (JNK) was also activated within the same time frame. Treatment of cardiac myocytes with extracellular ceramides also activated JNK. Pretreating cells with antioxidants quenched sphingomyelinase activation, ceramide accumulation, and JNK activation. Ceramide did not accumulate in reoxygenated nonmuscle fibroblasts, and JNK was not activated by reoxygenation in these cells. The results identify neutral sphingomyelinase activation as one of the earliest responses of cardiac myocytes to the redox stress imposed by hypoxia-reoxygenation. The results are consistent with a pathway of ceramide-mediated activation of JNK.