Short-chain L-3-hydroxy-2-methylacyl-CoA dehydrogenase (SC-HMAD), a soluble mitochondrial enzyme, was purified 6000-fold from rat Liver in 6% yield by a six-step purification procedure. The purified enzyme was homogenous as judged by gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular mass of this protein was estimated to be 28 kDa under denaturing conditions. Under nondenaturing conditions, the enzyme behaved on Sephacryl S-200 like serum albumin with a molecular mass of 66 kDa. Thus, SC-HMAD seems to be a dimer composed of two, most likely identical 28-kDa subunits. Immunoblotting with antibodies to pig heart L-3-hydroxyacyl-CoA dehydrogenase (HAD) (EC 1.1.1.35) revealed that SC-HMAD and HAD are immunologically unrelated proteins. SC-HMAD, but not HAD, catalyzes the NAD⁺-dependent dehydrogenation of L-3-hydroxy-2-methybutyryl-CoA, a metabolite of isoleucine, to 2-methylacetoacetyl-CoA. Relative activities with 3-hydroxy-2-methylacyl-CoA thioesters having acyl chains with 4, 5, 10, and 16 carbon atoms are 88, 100, 16, and 0%, respectively. Unbranched 3-hydroxyacyl-CoA thioesters are also substrates of SC-HMAD, although poorer ones as evidenced by apparent K_m values of 5 and 19 μM for L-3-hydroxy-2-methylbutyryl-CoA and L-3-hydroxybutyryl-CoA, respectively. Maximal velocities observed with these two substrates were similar. It is concluded that SC-HMAD catalyzes the second dehydrogenation step during the β-oxidation of the isoleucine metabolite 2-methylbutyryl-CoA. This enzyme may also be involved in the β-oxidation of natural and xenobiotic branched chain carboxylic acids.