Carnitine palmitoyltransferase-I (CPT-I) is considered to be a rate-limiting step in the mitochondrial translocation and β-oxidation of free fatty acids. Based on in vitro studies, the rest and exercise concentrations of its inhibitor, malonyl-CoA (M-CoA), should significantly inhibit CPT-I activity and preclude the increase in free fatty acid use during exercise. We investigated the effects of substrate concentration, pH, temperature, and potential regulators of CPT-I activity in vitro in the absence and presence of physiological M-CoA concentrations. CPT-I activity displayed Michaelis-Menten kinetics with increasing concentrations of palmitoyl-CoA and carnitine. CPT-I activity was not different between temperatures (34, 37 and 40°C), but was inhibited at pH 6.8 and 6.4, compared to 7.0. CPT-I was strongly inhibited (>80%) by M-CoA concentrations above 2μM (IC50 = 0.22 ± 0.04 uM). CPT-I activity was not inhibited by acetylcarnitine (0 - 50 μM) or acetyl-CoA (0 - 50 μM) and was only slightly (∼20%) inhibited by CoASH (0 - 50 μM). Finally, M-CoA inhibition was not relieved by the addition of acetyl-CoA, CoASH, or acetylcarnitine in concentrations that simulated resting and exercise conditions. Present results do not explain the relief of M-CoA inhibition of CPT-I activity that must occur with exercise.