Abstract The Escherichia coli α-ketoglutarate dehydrogenase complex contains a core, consisting of dihydrolipoyl transsuccinylase, to which α-ketoglutarate dehydrogenase and dihydrolipoyl dehydrogenase (a flavoprotein) are joined by noncovalent bonds. The molecular weights of the three enzymes isolated from the complex are: α-ketoglutarate dehydrogenase, about 190,000; dihydrolipoyl transsuccinylase, about 1,000,-000; and dihydrolipoyl dehydrogenase, about 112,000. Both the isolated α-ketoglutarate dehydrogenase and the flavoprotein exist in solution at pH 7.0 as stable dimers that contain two apparently identical polypeptide chains. Although the transsuccinylase core consists of 24 very similar polypeptide chains, only 12 of these chains contain a covalently bound lipoyl moiety. The subunit composition of the native α-ketoglutarate dehydrogenase complex is about 12 α-ketoglutarate dehydrogenase chains, 24 transsuccinylase chains, and about 12 flavoprotein chains. Reconstitution experiments indicate that the transsuccinylase can bind about six α-ketoglutarate dehydrogenase dimers (i.e. 12 chains) and about 18 flavoprotein dimers (i.e. 36 chains). However, nearly maximum activity is obtained when only about six flavoprotein dimers (i.e. about 12 chains) are bound. A possible explanation of these findings is that a systematic perturbation of the structure of the transsuccinylase has reduced its symmetry from 432 to 23 so that instead of one set of 24 equivalent sites for each ligand the transsuccinylase has two sets of 12 equivalent sites. The perturbation need not be a large one, and the binding energies of the nonequivalent sites could be different.