The Drosophila melanogaster genome contains only one CPT1 gene (Jackson, V. N., Cameron, J. M., Zammit, V. A., and Price, N. T. (1999) Biochem. J. 341, 483-489). We have now extended our original observation to all insect genomes that have been sequenced, suggesting that a single CPT1 gene is a universal feature of insect genomes. We hypothesized that insects may be able to generate kinetically distinct variants by alternative splicing of their single CPT1 gene. Analysis of the insect genomes revealed that (a) the single CPT1 gene in each and every insect genome contains two alternative exons and (ii) in all cases, the putative alternative splicing site occurs within a small region corresponding to 21 amino acid residues that are known to be essential for the binding of substrates and of malonyl-CoA in mammalian CPT1A.Weperformed PCR analyses of mRNA from different Drosophila tissues; both of the anticipated splice variants of CPT1mRNAwere found to be expressed in all of the tissues tested (both in larvae and adults), with the expression level for one of the splice variants being significantly different between flight muscle and the fat body of adult Drosophila. Heterologous expression of the full-length cDNAs corresponding to the two putative variants of Drosophila CPT1 in the yeast Pichia pastoris revealed two important differences between the properties of the two variants: (i) their affinity (K 0.5) for one of the substrates, palmitoyl-CoA, differed by 5-fold, and (ii) the sensitivity to inhibition by malonyl-CoA at fixed, higher palmitoyl-CoA concentrations was 2-fold different and associated with different kinetics of inhibition. These data indicate that alternative splicing that specifically affects a structurally crucial region of the protein is an important mechanism through which functional diversity of CPT1 kinetics is generated from the single gene that occurs in insects.