Whole-body hyperthermia induces heat shock proteins (HSPs) which confer cardioprotection. Several opioid receptor subtypes are expressed in the heart and are linked to cardioprotection; however, no one has attempted to link the protection elicited by heat stress to opioids. Therefore, we investigated the effect of an opiate receptor antagonist, naloxone, on heat stress (HS)-induced cardioprotection. Anesthetized Sprague-Dawley rats were subjected to HS (42°C for 20 minutes) with and without naloxone pretreatment and allowed to recover for 48 hours. They then underwent 30 minutes of ischemia followed by 2 hours of reperfusion. An acute HS group was given an IV bolus of naloxone (3mg/kg) 10 minutes prior to index ischemia. Infarct size, expressed as a percent of the area at risk (IS/AAR), was determined. The right heart was excised for analysis of HSP content by Western blot. Heat shocked rats showed significant reductions in IS/AAR versus control (16±3 vs. 58±4, p<0.001). Pretreatment with naloxone prior to HS attenuated protective effects in a dose dependent fashion, with significant attenuation of protection occurring at 15mg/kg naloxone vs. heat shock (42±6 vs. 16±3, p<0.001). Acute treatment with naloxone (3mg/kg) 48 hours after recovery from heat stress also significantly attenuated the delayed protective effect (47±4 vs. 16±3, p<0.001). No difference was seen in the level of HSP 70 induced in the different groups. We conclude that heat shock-induced cardioprotection can be attenuated by naloxone, an opiate receptor antagonist, without reducing the levels of certain HSPs. These results suggest there may be a link between the endogenous release of opioids and heat stress that mediates cardioprotection, though strain specific effects may exist.