We tested the hypothesis that exposure to altitude decreases reliance on free fatty acids (FFA) as substrates and increases dependency on blood glucose. Therefore, the effects of exercise, hypobaric hypoxia, and altitude acclimatization on FFA, glycerol and net glucose uptake and release [= 2(leg blood flow)(arteriovenous concentration)] and on fatty acid (FA) consumption by the legs (= 3 x glycerol release + FFA uptake) were measured. Because sympathetic responses have been implicated, we utilized nonspecific beta-blockade and observed responses to exercise, altitude, and altitude acclimatization. We studied six healthy beta-blocked men (beta) and five matched controls (C) during rest and cycle ergometry exercise (88 W) at 49% of sea-level (SL) peak O2 uptake at the same absolute power output on acute altitude exposure (A1; barometric pressure = 430 Torr) and after 3 wk of chronic altitude exposure to 4,300 m (A2). During exercise at SL, FA consumption rates increased (P < 0.05). On arrival at 4,300 m, resting leg FFA uptake and FA consumption rates were not significantly different from those at SL. However, after acclimatization to altitude, at rest leg FA consumption decreased to essentially zero in both C and beta groups. During exercise to altitude after acclimatization, leg FA consumption increased significantly, but values were less than at SL or A1 (P < 0.05), whereas glucose uptake increased relative to SL values. Furthermore, beta-blockade significantly increased glucose uptake relative to control. We conclude that 1) chronic altitude exposure decreases leg FA consumption during rest and exercise; 2) relative to SL FFA uptake decreases while glucose uptake increases during exercise at altitude; and 3) beta-blockade potentiates these effects.