The σS and σ70 subunits of Escherichia coli RNA polymerase recognize very similar promoter sequences. Therefore, many promoters can be activated by both holoenzymes in vitro. The same promoters, however, often exhibit distinct sigma factor selectivity in vivo. It has been shown that high salt conditions, reduced negative supercoiling and the formation of complex nucleoprotein structures in a promoter region can contribute to or even generate σS selectivity. Here, we characterize the first positively acting σS-selective feature in the promoter sequence itself. Using the σS-dependent csiD promoter as a model system, we demonstrate that C and T at the −13 and −14 positions, respectively, result in strongest expression. We provide allele-specific suppression data indicating that these nucleotides are contacted by K173 in region 2.5 of σS. In contrast, σ70, which features a glutamate at the corresponding position (E458), as well as the σS(K173E) variant, exhibit a preference for a G(−13). C(−13) is highly conserved in σS-dependent promoters, and additional data with the osmY promoter demonstrate that the K173/C(−13) interaction is of general importance. In conclusion, our data demonstrate an important role for region 2.5 in σS in transcription initiation. Moreover, we propose a consensus sequence for a σS-selective promoter and discuss its emergence and functional properties from an evolutionary point of view.