Branched chain ketoacid dehydrogenase has been isolated and purified from bovine liver mitochondria. This multienzyme complex oxidatively decarboxylates the ketoacid derivatives of valine, leucine, and isoleucine. The substrates competitively inhibit one another showing Ki values in agreement with their determined Km values of 8 micromoles Thiamin pyrophosphate, the first cofactor in the reaction sequence has been shown to induce a conformational change in the complex which is thought to be responsible for the stabilization against heat and proteolytic inactivation observed to be provided by the binding of this cofactor. CoASH and NAD, the other cofactors of the reaction, bind less tightly than thiamin pyrophosphate and provide no protection against inactivation as tested thus far. Studies with sulfhydryl reagents support the involvement of covalently bound lipoic acid in the reaction mechanism. Antibodies have been raised in rabbits to the active complex and to the liopamide oxidoreductase component of the complex. Rabbits are currently being sensitized to the other protein components of the complex. These will be used to investigate the organization of the branched chain ketoacid dehydrogenase complex in the mitochondrial inner membrane.