We hypothesized that reliance on lactate as a means of energy distribution is higher after a prolonged period of acclimatization (9 wk) than it is at sea level due to a higher lactate R_a and disposal from active skeletal muscle. To evaluate this hypothesis, six Danish lowlanders (25 ± 2 yr) were studied at rest and during 20 min of bicycle exercise at 146 W at sea level (SL) and after 9 wk of acclimatization to 5,260 m (Alt). Whole body glucose R_a was similar at SL and Alt at rest and during exercise. Lactate R_a was also similar for the two conditions at rest; however, during exercise, lactate R_a was substantially lower at SL (65 µmol · min¹ · kg body wt¹) than it was at Alt (150 µmol · min¹ · kg body wt¹) at the same exercise intensity. During exercise, net lactate release was ~6-fold at Alt compared with SL, and related to this, tracer-calculated leg lactate uptake and release were both 3- or 4-fold higher at Alt compared with SL. The contribution of the two legs to glucose disposal was similar at SL and Alt; however, the contribution of the two legs to lactate R_a was significantly lower at rest and during exercise at SL (27 and 81%) than it was at Alt (45 and 123%). In conclusion, at rest and during exercise at the same absolute workload, CHO and blood glucose utilization were similar at SL and at Alt. Leg net lactate release was severalfold higher, and the contribution of leg lactate release to whole body lactate R_a was higher at Alt compared with SL. During exercise, the relative contribution of lactate oxidation to whole body CHO oxidation was substantially higher at Alt compared with SL as a result of increased uptake and subsequent oxidation of lactate by the active skeletal muscles.