High-altitude acclimation alters lipid metabolism during exercise, but it is unknown whether this involves changes in rates of lipolysis or reesterification, which form the triacylglycerol/fatty acid (TAG/FA) cycle. We combined indirect calorimetry with [2-3H]glycerol and [1-14C]palmitate infusions to simultaneously measure total lipid oxidation, lipolysis, and rate of appearance (Ra) of nonesterified fatty acids (NEFA) in high-altitude-acclimated (HA) rats exercising at 60% maximal O2 uptake (V˙o 2 max). During exercise, relative total lipid oxidation (%V˙o 2) equaled sea-level control (SL) values; however, acclimation greatly stimulated lipolysis (+75%) but had no effect on Ra NEFA. As a result, TAG/FA cycling increased (+119%), due solely to an increase in recycling (+144%) within adipocytes. There was no change in either group in these variables with the transition from rest to exercise. We conclude that, in HA, 1) acclimation is a potent stimulator of lipolysis; 2) rats do not modify TAG/FA cycling with the transition to exercise; and 3) in normoxia, HA and SL derive the same fraction of their total energy from lipids and carbohydrates.