The present study examined the sensitivity of carnitine palmitoyltransferase I (CPT I) activity to its inhibitor malonyl-CoA (M-CoA), and simulated metabolic conditions of rest and exercise, in aerobically trained and untrained humans. Maximal CPT I activity was measured in mitochondria isolated from resting human skeletal muscle. Mean CPT I activity was 492.8 ± 72.8 and 260.8 ± 33.6 μmol ⋅ min−1 ⋅ kg wet muscle−1 in trained and untrained subjects, respectively (pH 7.0, 37°C). The sensitivity to M-CoA was greater in trained muscle; the IC50 for M-CoA was 0.17 ± 0.04 and 0.49 ± 0.17 μM in trained and untrained muscle, respectively. The presence of acetyl-CoA, free coenzyme A (CoASH), and acetylcarnitine, in concentrations simulating rest and exercise conditions did not release the M-CoA-induced inhibition of CPT I activity. However, CPT I activity was reduced at pH 6.8 vs. pH 7.0 in both trained and untrained muscle in the presence of physiological concentrations of M-CoA. The results of this study indicate that aerobic training is associated with an increase in the sensitivity of CPT I to M-CoA. Accumulations of acetyl-CoA, CoASH, and acetylcarnitine do not counteract the M-CoA-induced inhibition of CPT I activity. However, small decreases in pH produce large reductions in the activity of CPT I and may contribute to the decrease in fat metabolism that occurs during moderate and intense aerobic exercise intensities.