Transport of long-chain fatty acids from the cytosol to the mitochondria1 matrix for P-oxidation involves the conversion of long-chain fatty acyl-CoA to acylcamitines in the presence of L-carnitine by carnitine palrnitoyltransferase I (CPTI), translocation across the inner mitochondria1 membrane by the carnitine carrier and reconversion to long-chain fatty acyl-CoA by carnitine palrnitoyltransferase 11 (CPTII). As an enzyme that catalyzes the first step in fatty acid oxidation, CPTI is regulated by its physiological inhibitor, malonyl-CoA, and by hormonal and dietary factors. In this study, we identified specific conserved amino acid residues important for activity and malonyl-CoA sensitivity in L-CPTI, M-CPTI and CPTII