Escherichia coli RNA polymerase associated with the σ54 factor (RNAP·σ54) is a holoenzyme form that transcribes a special class of promoters not recognized by the standard RNA polymerase·σ70 com plex. Promoters for RNAP·σ54 vary in their overall ‘strength’ and show differences in their response to the presence of DNA curvature between enhancer and promoter. In order to examine whether these effects are related to the promoter affinity, we have determined the equilibrium dissociation constant Kd for the binding of RNAP·σ54 to the three promoters glnAp2, nifH and nifL. Binding studies were conducted by monitoring the changes in fluorescence anisotropy upon titrating RNAP·σ54 to carboxyrhodamine-labeled DNA duplexes. For the glnAp2 and nifH promoters similar values of Kd = 0.94 ± 0.55 nM and Kd = 0.85 ± 0.30 nM were determined at physiological ionic strength, while the nifL promoter displayed a significantly weaker affinity with Kd = 8.5 ± 1.9 nM. The logarithmic dependence of Kd on the ionic strength I was –Δlog(Kd)/Δlog(I) = 6.1 ± 0.5 for the glnAp2, 5.2 ± 1.2 for the nifH and 2.1 ± 0.1 for the nifL promoter. This suggests that the polymerase can form fewer ion pairs with the nifL promoter, which would account for its weaker binding affinity.