This study determined the role of body temperature during exercise on Cytochrome c oxidase activity (CYTOX), a marker of mitochondrial content, and mitochondrial heat shock protein (mtHSP70), which is required for import of nuclear-coded pre-proteins. Male, 10-week-old, Sprague-Dawley rats exercised identically for 9 weeks in ambient temperatures of: 23°C (n=10); 8°C with wetted fur (n=8); and 4°C with wetted fur and fan (n=7). These conditions maintained exercising core temperature (T_c) at 40.4°C, 39.2°C, or 38.0°C (resting temperature), respectively. During weeks 3-9 the exercisers ran 5 days/wk up a 6% grade at 20 m/min for 60 mactivity in T_c=38.0° (83.5±5.5 µAtoms O/min/g wet wt) was greater than all other groups (p<0.05), exceeding sedentary (n=7) by 73.2%. T_c=40.4° and T_c=39.2° also were higher than sedentary by 22.4% and 37.4%, respectively (p<0.05). Quantification of CYTOX content verified that the increased activity was due to an increase in protein content. In EDL, a non-active muscle, CYTOX was not elevated in T_c=38.0°. mtHSP70 was significantly elevated in gastrocnemius of T_c=38.0° compared to sedentary (P<0.05), but was not elevated in EDL (P>0.05). The data indicate that decreasing exercise core temperature enhances mitochondrial biogenesis.