A mutant of Escherichia coli deficient in dihydrolipoyl dehydrogenase (DHL) activity has been isolated and its characteristics have been studied. The activities of the pyruvic dehydrogenase (PDC) and alpha-ketoglutaric dehydrogenase complexes (KDC) are not present in extracts of the mutant unless purified dihydrolipoyl dehydrogenase is added. Experiments with antiserum to DHL have shown that cross-reacting material exists in mutant extracts. This suggests that the dihydrolipoyl dehydrogenase mutation (dhl(-)) is a missense structural mutation. The mutation maps very close to, if not adjacent to, the ace loci, and is not linked to the suc loci. This means the dhl locus is grouped with the genes for the other components of the PDC and not with the genes for KDC. The mutation is also transducible into prototrophic strains, demonstrating that no prior mutation is necessary for the DHL activity deficiency to exist. This evidence is consistent with the idea that there is only one gene for DHL and is supported by previous biochemical studies which have shown that DHL preparations from either enzyme complex are electrophoretically and immunochemically indistinguishable. Possible mechanisms for the genetic and metabolic control of DHL, PDC, and KDC are discussed.