Pig and rat liver carnitine palmitoyltransferase I (L-CPTI) share common Km values for palmitoyl-CoA and carnitine. However, they differ widely in their sensitivity to malonyl-CoA inhibition. Thus, pig L-CPTI has an IC50 for malonyl-CoA of 141 nM, while that of rat L-CPTI is 2 M. Using chimeras between rat L-CPTI and pig L-CPTI, we show that the entire C-terminal region behaves as a single domain, which dictates the overall malonyl-CoA sensitivity of this enzyme. The degree of malonyl-CoA sensitivity is determined by the structure adopted by this domain. Using deletion mutation analysis, we show that malonyl-CoA sensitivity also depends on the interaction of this single domain with the first 18 N-terminal amino acid residues. We conclude that pig and rat L-CPTI have different malonyl-CoA sensitivity, because the first 18 N-terminal amino acid residues interact differently with the C-terminal domain. This is the first study that describes how interactions between the carboxyl and amino terminal regions can determine the malonyl-CoA sensitivity of L-CPTI enzymes using active C-terminal chimeras.