We have previously shown that heat shock protein 70 (HSP70) markedly inhibits H(2)O(2)-induced apoptosis in mouse C2C12 myogenic cells by reducing the release of Smac. However, the molecular mechanism by which HSP70 interferes with Smac release during oxidative stress-induced apoptosis is not understood. In the current study, we showed that HSP70 increased the stability of Bcl-2 during oxidative stress. An antisense phosphorothioate oligonucleotide against Bcl-2 caused selective inhibition of Bcl-2 protein expression induced by HSP70 and significantly attenuated HSP70-mediated cell protection against H(2)O(2)-induced release of Smac and apoptosis. Taken together, our results indicate that there are important relationships among HSP70, Bcl-2, release of Smac, and induction of apoptosis by oxidative stress.