Unsaturated fatty acids play an important role in the prevention of human diseases such as diabetes, obesity, cancer, and neurodegeneration. However, their oxidation in vivo by acyl-CoA dehydrogenases (ACADs) that catalyze the first step of each cycle of mitochondrial fatty acid -oxidation is not entirely understood. Recently, a novel ACAD (ACAD-9) of unknown function that is highly homologous to human very-long-chain acyl-CoA dehydrogenase was identified by large-scale random sequencing. To characterize its enzymatic role, we have expressed ACAD-9 in Escherichia coli, purified it, and determined its pattern of substrate utilization. The N terminus of the mature form of the enzyme was identified by in vitro mitochondrial import studies of precursor protein. A 37-amino acid leader peptide was cleaved sequentially by two mitochondrial peptidases to yield a predicted molecular mass of 65 kDa for the mature subunit. Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane. Gel filtration analysis indicated that, like verylong-chain acyl-CoA dehydrogenase, ACAD-9 is a dimer, in contrast to the other known ACADs, which are tetramers. Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA). These results suggest a previously unrecognized role for ACAD-9 in the mitochondrial -oxidation of long-chain unsaturated fatty acids. Because of the substrate specificity and abundance of ACAD-9 in brain, we speculate that it may play a role in the turnover of lipid membrane unsaturated fatty acids that are essential for membrane integrity and structure.Unsaturated fatty acids are the most abundant form of stored fat in the human body and are vital for all living organisms. In addition to their role as an energy source, they are integral constituents of cell membranes, playing a role in membrane fluidity, cell signaling, and membrane integrity (1). Numerous beneficial physiologic effects have been attributed to unsaturated fatty acids, including protection from obesity, diabetes, cancer, and atherosclerosis (2, 3). Utilization of unsaturated fatty acids requires mitochondrial -oxidation. Unsaturated fatty acids are more efficiently oxidized than long-chain saturated fatty acids in humans (4); however, the enzymes responsible for their catabolism have not been elucidated in their entirety. The observation that the initial cycles of -oxidation of long-chain unsaturated fatty acids (oleic acid (cis-9-C18:1) 5 and linoleic acid (cis-9,cis-12-C18:2)) occur in the absence of activity of very-long-chain acyl-CoA dehydrogenase (VLCAD) suggested the presence of an additional enzyme or alternate pathway (5). In contrast, the enzymatic -oxidation pathway for the saturated acyl-CoA esters is well described (6). The first step of fatty acid -oxidation is catalyzed by the acyl-CoA dehydrogenases (ACADs; EC 1.3.99.3), a family of mitochondrial enzymes with distinct substrate specificities (7). VLCAD and long-chain (...