Oxidative stress may cause apoptosis of cardiomyocytes in ischemia-reperfused myocardium, and heat shock pretreatment is thought to be protective against ischemic injury when cardiac myocytes are subjected to ischemia or simulated ischemia. However, the detailed mechanisms responsible for the protective effect of heat shock pretreatment are currently unclear. The aim of this study was to determine whether heat shock pretreatment exerts a protective effect against hydrogen peroxide(H₂O₂)–induced apoptotic cell death in neonatal rat cardiomyocytes and C₂C₁₂ myogenic cells and whether such protection is associated with decreased release of second mitochondria-derived activator of caspase–direct IAP binding protein with low pI (where IAP is inhibitor of apoptosis protein) (Smac/DIABLO) from mitochondria and the activation of caspase-9 and caspase-3. After heat shock pretreatment (42 ± 0.3°C for 1 hour, recovery for 12 hours), cardiomyocytes and C₂C₁₂ myogenic cells were exposed to H₂O₂ (0.5 mmol/L) for 6, 12, 24, and 36 hours. Apoptosis was evaluated by Hoechst 33258 staining and DNA laddering. Caspase-9 and caspase-3 activities were assayed by caspase colorimetric assay kit and Western analysis. Inducible heat shock proteins (Hsp) were detected using Western analysis. The release of Smac/DIABLO from mitochondria to cytoplasm was observed by Western blot and indirect immunofluorescence analysis. (1) H₂O₂ (0.5 mmol/L) exposure induced apoptosis in neonatal rat cardiomyocytes and C₂C₁₂ myogenic cells, with a marked release of Smac/DIABLO from mitochondria into cytoplasm and activation of caspase-9 and caspase-3, (2) heat shock pretreatment induced expression of Hsp70, Hsp90, and αB-crystallin and inhibited H₂O₂-mediated Smac/DIABLO release from mitochondria, the activation of caspase-9, caspase-3, and subsequent apoptosis. H₂O₂ can induce the release of Smac/DIABLO from mitochondria and apoptosis in cardiomyocytes and C₂C₁₂ myogenic cells. Heat shock pretreatment protects the cells against H₂O₂-induced apoptosis, and its mechanism appears to involve the inhibition of Smac release from mitochondria.