Relative exercise intensity (or %maximum O2consumption,V˙o2 max) controls fuel selection at sea level (SL) and after high-altitude acclimation (HA) in rats. In this context we used indirect calorimetry, [1-14C]palmitate infusions, and muscle triacylglycerol (TAG) measurements to determine 1) total lipid oxidation, 2) the relationship between circulatory nonesterified fatty acid (NEFA) flux and concentration, and 3) muscle TAG depletion after exercise in HA-acclimated rats. Aerobic capacity is decreased in trained rats after 10 wk of acclimation. Both SL and HA showed the same relative use of lipids at 60% [62 ± 5% (HA) and 61 ± 3% (SL) of O2consumption (V˙o2)] and 80% [46 ± 6% (HA) and 47 ± 5% (SL) ofV˙o2] of their respective V˙o2 max. At 60% V˙o2 max, plasma [NEFA] were higher in HA, but rate of appearance was essentially the same in both groups (at 30 min, 38 ± 9 vs. 49 ± 6 μmol ⋅ kg−1⋅ min−1in HA and SL, respectively). At this intensity SL showed no significant decrease in muscle TAG, but in HA it decreased by 64% in soleus and by 90% in red gastrocnemius. We conclude that 1) the relative contributions of total lipid are the same in SL and HA, contrary to differences in [NEFA], because the relationship between flux rate and [NEFA] is modified after acclimation, and 2) muscle TAG may play a more important role at HA.