The protein nitrogen regulator I (NRI)-phosphate is known to activate the initiation of transcription of the Escherichia coli glnA gene. This activation is facilitated by the binding of the protein to NRI-specific sites located upstream of the sigma 54-dependent glnA promoter. To determine whether binding of NRI-phosphate to upstream sites is sufficient for activation, we placed several promoters not normally activated by NRI-phosphate downstream of NRI binding sites and measured activation in intact cells and in an in vitro transcription system. We found that the sigma 70-dependent lac promoter was not activated, that the sigma 54-dependent Klebsiella pneumoniae nifH promoter was weakly activated, and that a nifH promoter altered in the RNA polymerase binding site was almost as well activated as the glnA promoter. We conclude that the sensitivity of the susceptible promoter depends on the presence of NRI binding sites, but that the presence of bound NRI-phosphate upstream of a promoter is not sufficient for activation of transcription by RNA polymerase. This activation is determined by the structure of the RNA polymerase binding site. We suggest that sigma 54-but not sigma 70-dependent promoters are susceptible to activation by NRI-phosphate and that the nucleotide sequence of the sigma 54-RNA polymerase binding site is an important determinant of the efficiency of activation.