Activating Region 1 of Escherichia coli FNR protein is proposed to interact directly with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) during transcription activation at FNR-regulated promoters. Using an alphaCTD alanine scan mutant library, we have identified the residues of alphaCTD that are important for FNR-dependent transcription activation. Residues Asp-305, Gly-315, Arg-317, Leu-318 and Asp-319 are proposed to be the key residues in the contact site on alphaCTD for Activating Region 1 of FNR. In previous work, it had been shown that Activating Region 1 of FNR is a large surface-exposed patch and that the two crucial amino acid residues are Thr-118 and Ser-187. In this work, we have constructed Arg-118 FNR and Arg-187 FNR and shown that both FNR derivatives are defective in transcription activation. However, the activity of FNR carrying Arg-118 can be partially restored by substitutions of Lys-304 in alphaCTD. Similarly, the activity of FNR carrying Arg-187 can be partially restored by substitutions of Arg-317 or Leu-318 in alphaCTD. The specificity of the restoration suggests that, during transcription activation by FNR, the side-chain of residue 118 in Activating Region 1 of FNR is located close to Lys-304 and Asp-305 in alphaCTD. Similarly, the side-chain of residue 187 in Activating Region 1 of FNR is located close to Arg-317 and Leu-318 in alphaCTD. These results can be used to model the interface between Activating Region 1 of FNR and its contact target in alphaCTD, and permit comparison of this interface with the interface between Activating Region 1 of the related transcription activator, CRP and alphaCTD.