Hypoxia impairs metabolic functions by decreasing activity and expression of ATP-consuming processes. To separate hypoxia from systemic effects, we tested whether hypoxia at high altitude affects basal and PMA-stimulated leukocyte metabolism and how this compares to acute (15 min) and 24 h of in vitro hypoxia. Leukocytes were prepared at low altitude and ∼24 h after arrival at 4559 m. Mitochondrial oxygen consumption (JO₂) was measured by respirometry, oxygen radicals by electron spin resonance spectroscopy, both at a Po₂ = 100 mmHg (JO₂,₁₀₀) and 20 mmHg (JO₂,₂₀). Acute hypoxia of leukocytes decreased JO₂ at low altitude. Exposure to high altitude decreased JO₂,₁₀₀, whereas JO₂,₂₀ was not affected. Acute hypoxia of low-altitude samples decreased the activity of complexes I, II, and III. At high altitude, activity of complexes I and III were decreased when measured in normoxia. Stimulation of leukocytes with PMA increased JO₂,₁₀₀ at low (twofold) and high altitude (five-fold). At both locations, PMA-stimulated JO₂ was decreased by acute hypoxia. Basal and PMA-stimulated reactive oxygen species (ROS) production were unchanged at high altitude. Separate in vitro experiments performed at low altitude show that ∼75% of PMA-induced increase in JO₂ was due to increased extra-mitochondrial JO₂ (JO₂(,res); in the presence of rotenone and antimycin A). JO₂(,res) was doubled by PMA. Acute hypoxia decreased basal JO₂(,res) by ∼70% and PMA-stimulated JO₂(,res) by about 50% in cells cultured in normoxia and hypoxia (1.5% O₂; 24 h). Conversely, 24 h in vitro hypoxia decreased mitochondrial JO₂,₁₀₀ and JO₂,₂₀, extra-mitochondrial, basal, and PMA-stimulated JO₂ were not affected. These results show that 24 h of high altitude but not 24 h in vitro hypoxia decreased basal leukocyte metabolism, whereas PMA-induced JO₂ and ROS formation were not affected, indicating that prolonged high-altitude hypoxia impairs mitochondrial metabolism but does not impair respiratory burst. In contrast, acute hypoxia impairs respiratory burst at either altitude.