This study determined the role of body temperature during exercise on cytochrome-c oxidase (CytOx) activity, a marker of mitochondrial content, and mitochondrial heat shock protein 70 (mtHSP70), which is required for import of nuclear-coded preproteins. Male, 10-wk-old, Sprague-Dawley rats exercised identically for 9 wk 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, 39.2, or 38.0°C (resting temperature), respectively. During weeks 3-9, exercisers ran 5 days/wk up a 6% grade at 20 m/min for 60 min. Animals were housed at 23°C. Gastrocnemius CytOx activity in T_c=38.0°C (83.5 ± 5.5 µatoms O · min¹ · g wet wt¹) was greater than all other groups (P < 0.05), exceeding sedentary (n = 7) by 73.2%. T_c of 40.4 and 39.2°C 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 extensor digitorum longus, a nonactive muscle, CytOx was not elevated in T_c = 38.0°C. mtHSP70 was significantly elevated in gastrocnemius of T_c = 38.0°C compared with sedentary (P < 0.05) but was not elevated in extensor digitorum longus (P > 0.05). The data indicate that decreasing exercise T_c may enhance mitochondrial biogenesis and that mtHSP70 expression is not dependent on temperature.