Background - Previous studies in isolated cardiac myocytes have shown that tumor necrosis factor (TNF)-α provokes increased expression of 27- and 70-kD stress proteins as well as manganese superoxide dismutase, suggesting that TNF-α might play a role in mediating stress responses in the heart. Methods and Results - To determine whether TNF-α stimulation would protect isolated cardiac myocytes against environmental stress, myocyte cultures were pretreated with TNF-α for 12 hours and then subjected to continuous hypoxic injury (O2 content, 3 to 5 ppm) for 12 hours, followed by reoxygenation. Cell injury was assessed in terms of lactic dehydrogenase (LDH) release, 45Ca2+ uptake, and MTT metabolism. Pretreatment with TNF-α concentrations ≤50 U/mL significantly attenuated LDH release by hypoxic cells compared with diluent-treated hypoxic cells. Similar findings were observed with respect to 45Ca2+ uptake and MTT metabolism in TNF-α- pretreated cells that were subjected to prolonged hypoxia. To determine the mechanism for the TNF-α-induced protective effect, the cells were pretreated with heat shock protein (HSP) 72 antisense oligonucleotides. These studies showed that the protective effect of TNF-α was not inhibited by antisense oligonucleotides, despite use of a concentration of antisense that was sufficient to attenuate the TNF-α-induced increase in HSP 72 expression. Subsequent studies using mutated TNF ligands showed that activation of both types l and 2 TNF receptors was sufficient to confer a protective response in isolated cardiac myocytes through an as yet unknown pathway(s). Conclusions - Taken together, the above observations demonstrate that TNF-α pretreatment confers resistance to hypoxic stress in the adult cardiac myocyte through a novel mechanism that appears to be different from but not necessarily exclusive of the protective response conferred by HSP 72 expression.