Mitochondrial and peroxisomal enoyl-CoA hydratases were purified from rat liver. The mitochondrial enzyme, with a molecular weight of 161,000, was composed of 6 identical subunits. The molecular structure of the rat liver enzyme was very similar to that of the bovine liver enzyme. Acetoacetyl-CoA was a competitive inhibitor of the mitochondrial enzymes. The results of titration of the rat liver enzyme with acetoacetyl-CoA suggest that 3 subunits of the enzyme exhibit catalytic activity. The catalytic properties of the enzyme were studied. The peroxisomal enzyme was composed of one polypeptide with a molecular weight of 70,000-81,000. Some of the enzyme molecules were shown to be cleaved to two polypeptides in the cell by the following methods: amino acid analysis, peptide mapping and immunoprecipitin reaction. The catalytic properties of the peroxisomal enzyme were different from those of the mitochondrial enzyme. The peroxisomal enzyme is a bifunctional enzyme exhibiting 3-hydroxyacyl-CoA dehydrogenase activity. Studies on the titration with acetoacetyl-CoA, the effects of salts, SH titration and proteolytic inactivation suggest that the active centers for these two reactions are located at different sites.