The ResD-ResE signal transduction system is required for transcription of genes involved in aerobic and anaerobic respiration in Bacillus subtilis. Phosphorylated ResD (ResD approximately P) interacts with target DNA to activate transcription. A strong sequence similarity was detected in promoter regions of some ResD-controlled genes including fnr and resA. Single-base substitutions in the fnr and resA promoters were performed to determine a ResD-binding sequence. DNase I footprinting analysis indicated that ResD approximately P itself does not bind to fnr, but interaction of ResD approximately P with the C-terminal domain of the alpha subunit (alphaCTD) of RNA polymerase (RNAP) facilitates cooperative binding of ResD approximately P and RNAP, thereby increasing fnr transcription initiation. Consistent with this result, amino acid substitutions in alphaCTD, such as Y263A, K267A, A269I, or N290A, sharply reduced fnr transcription in vivo, and the K267A alphaCTD protein, unlike the wild-type protein, did not increase ResD approximately P binding to the fnr promoter. Amino acid residues of alphaCTD required for ResD-dependent fnr transcription, with the exception of N290, which may interact with DNA, constitute a distinct surface, suggesting that these residues likely interact with ResD approximately P.