The molecular basis of myocardial cell death in the ischemia-reperfused heart still remains to be clarified. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays an important role in stress-induced apoptosis. We studied ASK1^−/− mice to examine the role of ASK1 in ischemia–reperfusion injury. In the wild-type heart, ischemia–reperfusion resulted in necrotic injury, whereas infarct size was drastically reduced in the ASK1^−/− heart. The necrotic injury was not accompanied with any evidence of apoptosis such as an increase in TUNEL-positive cells, DNA fragmentation or the activation of caspase-3. ASK1^−/− cardiomyocytes were more resistant to H₂O₂- or Ca²⁺-induced apoptotic and non-apoptotic cell death compared with wild-type cells. These data suggest that ASK1 is involved in necrosis as well as apoptosis and that ASK1-dependent necrosis is likely to contribute to myocardial cell death in the ischemia-reperfused heart.