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Gastrocnemius subsarcolemmal and intermyofibrillar mitochondria were isolated from 5-week-old cold-acclimated and thermoneutral control ducklings. In vitro respiration (polarography) and ATP synthesis (bioluminescence) were determined at 25 o C. Subsarcolemmal mitochondria showed a higher state 4 respiration and lower respiratory control and ADP/O ratio in cold-acclimated than in thermoneutral ducklings. Palmitate decreased the rate of ATP synthesis in both mitochondrial populations to about 30% of maximal but failed to abolish this process even at high concentrations. It is suggested that both expensive ATP synthesis and increased ATP hydrolysis could contribute synergistically to muscle non-shivering thermogenesis in cold-acclimated ducklings.