The cDNA encoding rat very long-chain acyl-CoA synthetase (VLACS) was cloned, using degenerative primers synthesized according to the partial amino acid sequences of the peptide fragments of the purified rat liver enzyme. The longest cDNA insert was 2972 base pairs with a 1860-base pair open reading frame encoding 620 amino acids. The calculated molecular mass of 70,692 daltons was consistent with size of the purified enzyme. In Northern blot analysis, a single band was detected at the position of about 3 kilobases, corresponding to the size of the cloned cDNA. cDNA-directed expression in Escherichia coli resulted in accumulation of expressed protein, as an inclusion body. An antibody was raised using this expressed protein to characterize the cDNA and the enzyme. The subcellular localization of VLACS in peroxisomes and microsomes was demonstrated in Western blot analysis. The specific activity and the substrate specificity of the cDNA expressed enzyme in COS-1 cells were consistent with those of the purified rat enzyme. The predicted amino acid sequence of VLACS had a high sequence similarity to fatty acid transport protein (Schaffer, J. E., and Lodish, H. F. (1994) Cell 79, 427-436), and was considered to have domains for adenylation and thioester formation. The entire structure of VLACS was dissimilar to that of long-chain acyl-CoA synthetase (Suzuki, H., Kawarabayashi, Y., Kondo, Y., Abe, T., Nishikawa, K., Kimura, S., Hashimoto, T., and Yamamoto, T. (1990) J. Biol. Chem. 265, 8681-8685), except for the domains.