A number of environmental and metabolic stimuli rapidly induce the expression of several highly conserved proteins such as heat shock proteins (HSPs) or stress proteins. The purpose of this study was to investigate the effects of a single bout of submaximal exercise in varying ambient temperatures on cardiac and skeletal muscle. Adult male Sprague-Dawley rats were randomly placed in one of three ambient temperature groups; control (23°C), hot (41°C) and cool (11°C). Each exercise bout consisted of treadmill running at 17 m/min and 0% grade. Tissue HSP70 levels for all groups were determined using analysis of variance in two factorial design (2×3). Baseline rectal temperature was similar for all three groups. In the control and hot temperature groups, final rectal temperatures differed from the baseline values ( p <.05). The rectal temperature from the control/exercise group were 38.5±0.3°C at rest and 39.8±0.3°C at exhaustion, the hot/exercise group were 38.4±0.3°C at rest and 41.2±0.9°C at exhaustion and the cool/exercise group were 38.2±0.3°C at rest and 38.5±0.2°C at exhaustion. The running time was 102.0±39.5 min at the control/exercise group, 44.1±18.0 min at the hot/exercise group, and 55.4±11.9 min at the cool/exercise group. The level of soleus, cardiac and extensor digitorium longus (EDL) HSP70 in cool temperature does not change during a single bout of submaximal exercise. Whereas a single bout of submaximal exercise in hot and control ambient temperatures increases HSP70 accumulation in locomotor muscles, such as the soleus and cardiac, but not in the EDL tissue. This study shows that the changes of HSP70 level induced by a single bout of submaximal exercise at various ambient temperatures (control, hot and cool) depend on the rectal temperature.