We cloned a consensus DNA site for the Escherichia coli FNR protein at different locations upstream of the E. coli melR promoter. FNR can activate transcription initiation at the melR promoter when the FNR binding site is centered around 41, 61, 71, 82, and 92 bp upstream from the transcription start. The SF73 positive control amino acid substitution in FNR interfered with transcription activation by FNR in each case. In contrast, the GA85 positive control substitution reduced activation only at the promoter, where the FNR binding site is 41 bp upstream of the transcript start. The SF73 substitution appears to identify an activating region of FNR that is important for transcription activation at promoters that differ in architecture. Experiments with oriented heterodimers showed that this activating region is functional in the upstream subunit of the FNR dimer at the promoter where FNR binds around 41 bp from the transcript start and in the downstream subunit at the promoters where FNR binds farther upstream.