Two NAD-dependent dehydrogenases exhibiting activity with L-alanine were detected in crude extracts of Streptomyces aureofaciens. The dominant enzyme was found to be an alanine dehydrogenase with 6 or 4 subunits, each of M(r) 45,000. The minor enzyme exhibited high activities with branched-chain amino acids, suggesting it is a valine dehydrogenase. The results demonstrate that S. aureofaciens has only one alanine dehydrogenase, which is catalytically active in a six- or four-subunit structure.