Human carnitine palmitoyltransferase 1B (CPT1B) is a highly malonyl-CoA-sensitive enzyme (IC50=0.097 microm) and has a positive determinant (residues 18-28) of malonyl-CoA inhibition. By contrast, rat carnitine palmitoyltransferase 1A is less sensitive to malonyl-CoA inhibition (IC(50)=1.9 microm), and has both a positive (residues 1-18) and a negative (residues 18-28) determinant of its inhibition. Interestingly, pig CPT1B shows a low degree of malonyl-CoA sensitivity (IC(50)=0.804 microm). Here, we examined whether any additional molecular determinants affect malonyl-CoA inhibition of CPT1B. We show that the malonyl-CoA sensitivity of CPT1B is determined by the length (either 50 or 128 residues) of the N-terminal region constructed by recombining pig and human enzymes. We also show that the N-terminal region of pig CPT1B carries a single positive determinant of malonyl-CoA sensitivity, but that this is located between residues 1 and 18 of the N-terminal segment. Importantly, we found a single amino acid variation (D17E) relevant to malonyl-CoA sensitivity. Thus, Asp17 is specifically involved, under certain assay conditions, in the high malonyl-CoA sensitivity of the human enzyme, whereas the naturally occurring variation, Glu17, is responsible for both the low malonyl-CoA sensitivity and high carnitine affinity characteristics of the pig enzyme. This is the first demonstration that a single naturally occurring amino acid variation can alter CPT1B enzymatic properties.