Rat liver mitochondrial fatty acid oxidation multienzyme complex consists of 4 mol of the alpha-subunit and 4 mol of the beta-subunit, and has three enzyme activities of long chain enoyl-CoA hydratase, long chain 3-hydroxyacyl-CoA dehydrogenase, and long chain 3-ketoacyl-CoA thiolase. The following cDNA clones for the rat enzyme complex were isolated, sequenced, and expressed: 1) the 2,789-base pair (bp) cDNA clone had a 2,289-bp open reading frame encoding a 82,511-Da precursor and a 78,637-Da mature subunit. The deduced amino acid sequence of this subunit revealed that this cDNA encodes the alpha-subunit and had regions similar to the structure of rat mitochondrial enoyl-CoA hydratase and rat mitochondrial enoyl-CoA isomerase on the amino-terminal side, and a part similar to that of pig mitochondrial 3-hydroxyacyl-CoA dehydrogenase on the carboxyl-terminal side. Expression of this cDNA in COS-1 cells yielded a protein with long chain enoyl-CoA hydratase and long chain 3-hydroxyacyl-CoA dehydrogenase activities. 2) The 1,943-bp cDNA clone had a 1,425-bp open reading frame encoding a 51,413-Da precursor and a 47,583-Da mature subunit. A high similarity of the structure to 3-ketoacyl-CoA thiolases and acetoacetyl-CoA thiolases from various sources suggests that this clone encodes the beta-subunits. Expression of this cDNA in COS-1 cells yielded a protein with long chain 3-ketoacyl-CoA thiolase activity. By phylogenetic analysis of the deduced amino acid sequences of the alpha- and beta-subunits with those of other beta-oxidation enzymes, it was suggested that the alpha-subunit is a descendant of short chain enoyl-CoA hydratase and short chain 3-hydroxyacyl-CoA dehydrogenase while the beta-subunit first diverged from a common ancestor gene of the thiolase family.