Transcription from the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters requires the positive control protein NifA and the alternative sigma factor σ⁵⁴, encoded by the rpoN gene. Transcription from the K. pneumoniae nifH promoter is fully dependent upon NifA bound at the upstream activator sequence (UAS) whereas the R. meliloti nifH promoter can be efficiently activated in the absence of this sequence and can also be activated by a mutant form of NifA unable to bind the UAS. The in vivo interaction of RNA polymerase-σ⁵⁴ with these promoters was examined using dimethyl sulphate footprinting. The R. meliloti nifH promoter but not the K. pneumoniae nifH promoter showed σ⁵⁴-dependent methylation protection of guanine residues at −14, −25 and −26, the most conserved nucleotides characteristic of σ⁵⁴-dependent promoters. A mutant derivative of the K. pneumoniae nifH promoter bearing transitions at positions from − 15 to −17 showed σ⁵⁴-dependent methylation protection of guanines −13, −24 and −25. The enhanced interaction of the RNA polymerase-σ⁵⁴ with this mutant promoter correlates with its increased level of activation by a form of NifA unable to bind the UAS. Use of in vivo KMnO₄ footprinting to detect single-stranded pyrimidine residues and in vivo methylation protection demonstrated that the σ⁵⁴-dependent protection observed in the R. meliloti and mutant K. pneumoniae nifH promoter results from the formation of a closed promoter complex. The isomerization of the pre-existing closed complex to an open promoter form, as judged by the local denaturation of promoter DNA which rendered sequences from +5 to −10 reactive towards KMnO₄, was shown to be fully dependent on NifA. We propose a model in which the fidelity of activation of σ⁵⁴-dependent promoters relies on a weak activator-independent interaction of RNA polymerase-σ⁵⁴ with the promoter. A specific interaction of the appropriate activator with its respective UAS is then required for the positive control protein to facilitate open complex formation.