Expression of the Escherichia coli adaptive response genes (ada, aidB, and alkA) is regulated by the transcriptional activator, Ada. However, the interactions of RNA polymerase and Ada with these promoters differ. In this report we characterize the interactions of Ada, methylated Ada (meAda), and RNA polymerase at the alkA promoter and contrast these interactions with those characterized previously for the ada and aidB promoters. At the alkA promoter, we do not detect the RNA polymerase alpha subunit-mediated binary complex detected at the ada and aidB promoters. In the presence of either of these two activators, RNA polymerase protects the alkA core promoter, including the elements at -35 and -10, and is more efficient in transcription initiation in vitro. RNA polymerase holoenzyme containing the alpha subunit mutation R265A is severely impaired in Ada-independent basal alkA transcription, shows no activation by Ada or meAda, and fails to bind the alkA promoter in vitro. Binding of the purified wild type alpha subunit to alkA was not detected, but a complex of promoter DNA, Ada or meAda, and alpha was observed in gel shift assays. These observations suggest that both forms of Ada protein activate alkA transcription by enhancing RNA polymerase holoenzyme and alpha subunit binding.