Messenger RNA for 3-hydroxyacyl-CoA dehydrogenase, a mitochondrial matrix enzyme of fatty acid beta-oxidation, was purified from livers of di(2-ethylhexyl)phthalate-treated rats by immunoadsorption of hepatic free polysomes to fixed cells of Staphylococcus aureus and enrichment for poly(A)-rich RNA by oligo(dT)-cellulose chromatography. Plasmid cDNA was constructed from this poly(A)-rich RNA by a modification of the method of Okayama and Berg and was transformed into the Escherichia coli DH1 strain. Plasmids containing cDNA sequences coding for 3-hydroxyacyl-CoA dehydrogenase were screened by differential colony hybridization, and were identified by hybrid-arrested translation and hybrid-selected translation. Plasmid pHADH-1, which contains a 1400-base-pair insert, hybridized to rat 3-hydroxyacyl-CoA dehydrogenase mRNA with a length of 1700 bases. Determination of the dehydrogenase mRNA by in vitro translation and dot-blot analysis with the cDNA probe showed that the induction of the enzyme in rat liver by di(2-ethylhexyl)phthalate could be attributed to an increase in the mRNA concentration.