M yocardial ischemia markedly increases the expression of several members of the stress/heat shock protein (HSP) family, especially the inducible HSP70 isoforms. Increased expression of HSP70 has been shown to exert a protective effect against a lethal heat shock. We have examined the possibility of using this resistance to a lethal heat shock as a protective effect against an ischemic-like stress in vitro using a rat embryonic heart-derived cell line H9c2 (2-1). Myogenic cells in which the heat shock proteins have been induced by a previous heat shock are found to become resistant to a subsequent simulated ischemic stress. In addition, to address the question of how much does the presence of the HSP70 contribute to this protective effect, we have generated stably transfected cell lines overexpressing the human-inducible HSP70. Embryonal rat heart-derived H9c2(2-1) cells were used for this purpose. This stably transfected cell line was found to be significantly more resistant to an ischemic-like stress than control myogenic cells only expressing the selectable marker (neomycin) or the parental cell line H9c2(2-1). This finding implicates the inducible HSP70 protein as playing a major role in protecting cardiac cells against ischemic injury.