In Clostridium magnum strain Wo Bd P1 the formation of the enzyme components of the acetoin dehydrogenase enzyme system E1 (acetoin:2,6-dichlorophenolindophenol oxidoreductase Ao:DCPIP OR), E2 (dihydrolipoamide acetyltransferase DHLTA) and E3 (dihydrolipoamide dehydrogenase DHLDH) were induced during growth on acetoin. Ao:DCPIP OR was purified from acetoin-grown cells in two steps by chromatography on DEAE-Sephacel and on Mono Q HR. Native Ao:DCPIP OR exhibited a M(r) of 138,000; it consisted of two different subunits of M(r) alpha 38,500 and M(r) beta 34,000, and it occurred most probably in a tetrameric alpha 2 beta 2 structure. The N-terminal amino acid sequences of the alpha- and beta-subunits revealed homologies to the N-termini of the corresponding subunits of Ao:DCPIP OR from Pelobacter carbinolicus and from Alcaligenes eutrophus; furthermore, the N-terminus of the beta-subunit exhibited homologies to the N-termini of beta-subunits from different 2-oxo acid dehydrogenases.