Branched-chain α-keto acid dehydrogenase (BCKADH) was solubilized as an enzyme complex from rat liver mitochondria by sonic treatment. Dehydrogenase (E₁) and dihydrolipoyltransacylase (E₂) components of the complex were purified in an associated form and resolved into individual components in the presence of 1M NaCl, while lipoamide dehydrogenase (E₃) component was dissociated from the complex during purification. Analysis by gel electrophoresis in dodecyl sulfate revealed the E₁ comprised two different subunits with apparent molecular weights of 36, 000 and 45, 500, presumably in an equal molar ratio, while E₂ consisted of a single subunit with an apparent molecular weight of 51, 000. The BCKADH complex was reconstituted by combining E₁, E₂, and E₃, and the formation of the complex was confirmed by analysis by sucrose density gradient centrifugation. The reconstituted enzyme complex oxidized not only α-ketoisovalerate (KIV), α-ketoiso-caproate (KIC), and α-keto-β-methylvalerate (KMV), but also pyruvate and α-ketoglutarate. Apparent K_m values were 10-12 μM for the branched-chain α-keto acids, 2.2mM for pyruvate, and 2.5mM for α-ketoglutarate.