The induction of the heat shock response is accepted to be a protective response, reducing injury and improving cell survival. However, when inflammation precedes heat shock there is an unexpected increase in injury, known as the heat shock paradox, which is hypothesized to be a mechanism underlying multi-organ dysfunction. We hypothesized that the heat shock paradox would occur in adult cardiac myocytes and that heat shock factor (HSF)1 would contribute to injury. Heat shock (HS) at 42°C and TNF (10 ng/ml) were used as the HS and the inflammatory insult, respectively. The combination of TNF followed by HS (TNF/HS) caused the greatest amount of apoptosis in adult rat cardiac myocytes. TNF/HS resulted in an increase in heat shock protein (HSP) 60, compared to untreated cells, those receiving HS/TNF, or TNF alone. There was no increase in heme oxygenase 1 in any of the groups. HSP72 increased in all the groups, with the greatest levels with TNF/HS. NFκB activation was greatest with TNF/HS. Pretreatment with a DNA binding decoy for HSF1 prevented the increase in HSPs and decreased apoptosis in all groups. However, the increase in iNOS, seen in all treatment groups, was unaffected by the HSF1 binding decoy. We conclude that the heat shock paradox occurs in adult cardiac myocytes, that HSP60 is increased as part of the heat shock paradox, and that HSF1 activation contributes to injury.