To investigate the influence of heat stress on the regulation of skeletal muscle carbohydrate metabolism, six active, but not specifically trained, men performed 5 min of cycling at a power output eliciting 70% maximal O₂ uptake in either 20°C (Con) or 40°C (Heat) after 20 min of passive exposure to either environmental condition. Although muscle temperature (T_mu) was similar at rest when comparing trials, 20 min of passive exposure and 5 min of exercise increased (P < 0.05) T_mu in Heat compared with Con (37.5 ± 0.1 vs. 36.9 ± 0.1°C at 5 min for Heat and Con, respectively). Rectal temperature and plasma epinephrine were not different at rest, preexercise, or 5 min of exercise between trials. Although intramuscular glycogen phosphorylase and pyruvate dehydrogenase activity increased (P < 0.05) at the onset of exercise, there were no differences in the activities of these regulatory enzymes when comparing Heat with Con. Accordingly, glycogen use in the first 5 min of exercise was not different when comparing Heat with Con. Similarly, no differences in intramuscular concentrations of glucose 6-phosphate, lactate, pyruvate, acetyl-CoA, creatine, phosphocreatine, or ATP were observed at any time point when comparing Heat with Con. These results demonstrate that, whereas mild heat stress results in a small difference in contracting T_mu, it does not alter the activities of the key regulatory enzymes for carbohydrate metabolism or glycogen use at the onset of exercise, when plasma epinephrine levels are unaltered.