To investigate the regulation of intramuscular fuel selection, we measured the malonyl-CoA (M-CoA) content in human skeletal muscle at three exercise power outputs [35, 65, and 90% maximal rate of O2 consumption (V˙o 2 max)]. Four males and four females cycled for 10 min at one power output on three separate occasions with muscle biopsies sampled at rest and at 1 and 10 min. The respiratory exchange ratio was 0.84 ± 0.03, 0.92 ± 0.02, and >1.0 at 35, 65 and 90%V˙o 2 max, respectively. Muscle lactate content increased and phosphocreatine content decreased as a function of power output. Pyruvate dehydrogenase a activity increased from 0.40–0.64 mmol ⋅ kg wet muscle−1 ⋅ min−1at rest to 1.57 ± 0.28, 2.80 ± 0.41, and 3.28 ± 0.27 mmol ⋅ kg wet muscle−1 ⋅ min−1after 1 min of cycling at the three power outputs, respectively. Mean resting M-CoA contents were similar at all power outputs (1.85–1.98 μmol/kg dry muscle). During exercise at 35%V˙o 2 max, M-CoA decreased from rest at 1 min (1.85 ± 0.29 to 1.20 ± 0.12 μmol/kg dry muscle) but returned to rest level by 10 min (1.86 ± 0.25 μmol/kg dry muscle). M-CoA content did not decrease during cycling at 65%V˙o 2 max. At 90%V˙o 2 max, M-CoA did not increase despite significant acetyl-CoA accumulation (the substrate for M-CoA synthesis). The data suggest that a decrease in M-CoA content is not required for the increase in free fatty acid uptake and oxidation that occurs during exercise at 35 and 65%V˙o 2 max. Furthermore, M-CoA content does not increase during exercise at 90%V˙o 2 max and does not contribute to the lower rate of fat oxidation at this power output.