Previous studies have demonstrated that oxygen consumption and fat oxidation remain elevated in the postexercise period. The purpose of this study was to determine whether malonyl-CoA, an inhibitor of fatty acid oxidation, remains depressed in muscle after exercise. Rats were sprinted for 5 min (40 m/min, 5% grade) or run for 30 min (21 m/min, 15% grade). Red quadriceps malonyl-CoA returned to resting values by 90 min postexercise in the sprinting rats and remained significantly lower at least 90 min postexercise in the 30-min exercise group. AMP-activated protein kinase activity remained significantly elevated (P < 0.05) for 10 min after exercise in both groups. The most rapid rate of glycogen repletion was in the first 30 min postexercise. The respiratory exchange ratio decreased from a nonexercise value of 0.87 ± 0.01 to an average 0.82 ± 0.01 during the 90-min period after 30 min of exercise. Thus muscle malonyl-CoA remains depressed and fat oxidation is elevated for relatively prolonged periods after a single bout of exercise. This may allow fat oxidation to contribute more to muscle energy requirements, thus leaving more glucose for replenishment of muscle glycogen.