Two promoters required for expression of the ask-asd genes, encoding aspartokinase (AK) and aspartate-semialdehyde dehydrogenase (ASD), in Corynebacterium flavum N13, askP1 and askP2, have been identified by deletion analysis and S1 nuclease mapping. Transcription from askP1 initiates 35 and 38 bp upstream of the ask structural gene. A second promoter, askP2, lies within the ask coding region, upstream of the translation start site of the AK beta subunit and can direct the expression of AK beta and ASD. Western immunoblot analysis and heterologous expression in Escherichia coli demonstrate that two separate polypeptides, a 44.8-kDa alpha subunit and an 18.5-kDa beta subunit, are expressed from the C. flavum N13 ask gene from distinct, in-frame translation initiation sites. A second AK mutation, G345D, which reduces the sensitivity of AK to concerted feedback inhibition by threonine plus lysine, was identified.