Trifunctional protein deficiency, a typical mitochondrial long-chain fatty acid beta-oxidation defect, is caused by the abnormality of mitochondrial long-chain enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein consisting of four moles of alpha-subunit and four moles of beta-subunit. We cloned, sequenced, and expressed the following cDNAs for the alpha- and beta-subunits of human trifunctional protein. The 2,690-bp cDNA clone had a 2,289-bp open reading frame encoding a 82,958-Da precursor and a 78,969-Da mature subunit (alpha-subunit). Expression of this cDNA in mammalian cells yielded a polypeptide with the long-chain enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase activities. The 1,991-bp cDNA clone had a 1,422-bp open reading frame encoding a 51,293-Da precursor and a 47,484-Da mature subunit (beta-subunit). Expression of this cDNA in mammalian cells yielded a polypeptide with the long-chain 3-ketoacyl-CoA thiolase activity.