At high altitude (HA), carbohydrate (CHO) is thought to be the preferred fuel because of its higher yield of ATP per mole of O 2 . We used indirect calorimetry and
d-[6- 3 H]glucose infusions to determine total CHO and circulatory glucose utilization during exercise in HA-acclimated and sea level (SL) rats. We hypothesized that the percent contribution of CHO to total metabolism (V̇ O 2 ) is determined by exercise intensity relative to an aerobic maximum (% V̇ O 2 max ). HA rats run under hypoxia (F IO 2 = 0.12) showed a decrease in V̇ O 2 max compared with SL (67.55 ± 1.26 vs. 89.30 ± 1.23 ml kg −1 min −1 ). When exercised at 60% of their respective V̇ O 2 max , both groups showed the same relative use of CHO (38 ± 3% and 38 ± 5% of V̇ O 2 , at the beginning of exercise, in HA and SL, respectively). In both HA and SL, circulatory glucose accounted for ≈20% of V̇ O 2 , the balance was provided by muscle glycogen (≈18% of V̇ O 2 ). After 20 min at a higher intensity of 80% V̇ O 2 max , 54 ± 5% (HA) and 59 ± 4% (SL) of V̇ O 2 was accounted for by CHO. We conclude the following: ( i) the relative contributions of total CHO, circulatory glucose, and muscle glycogen do not increase after HA acclimation because the O 2 -saving advantage of CHO is outweighed by limited CHO stores; and ( ii) relative exercise intensity is the major determinant of metabolic fuel selection at HA, as well as at SL.