In eukaryotes, L-carnitine is involved in energy metabolism by facilitating β-oxidation of fatty acids. Carnitine acetyltransferases (CrAT) catalyze the reversible conversion of acetyl-CoA and carnitine to acetylcarnitine and free CoA. To redesign the specificity of rat CrAT toward its substrates, we mutated Met564. The M564G mutated CrAT showed higher activity toward longer chain acyl-CoAs: activity toward myiristoyl-CoA was 1250-fold higher than that of the wild-type CrAT, and lower activity toward its natural substrate, acetyl-CoA. Kinetic constants of the mutant CrAT showed modification in favor of longer acyl-CoAs as substrates. In the reverse case, mutation of the orthologous glycine (Gly553) to methionine in carnitine octanoyltransferase (COT) decreased activity toward its natural substrates, medium- and long-chain acyl-CoAs, and increased activity toward short-chain acyl-CoAs. Another CrAT mutant, M564A, was prepared and tested in the same way, with similar results. We conclude that Met564 blocks the entry of medium- and long-chain acyl-CoAs to the catalytic site of CrAT. Three-dimensional models of wild-type and mutated CrAT and COT support this hypothesis. We show for the first time that a single amino acid is able to determine the substrate specificity of CrAT and COT.