The rpoF gene of Escherichia coli codes for the RNA polymerase sigmaF (or sigma28) subunit, which is involved in transcription of the flagellar and chemotaxis genes. Both sigmaF and sigma70 (the major sigma subunit in growing cells) were overexpressed, purified to homogeneity, and compared with respect to activity and specificity. The affinity of sigmaF to core RNA polymerase (E) is higher than that of sigma70, as measured by gel filtration high-pressure liquid chromatography. In an in vitro transcription system, the holoenzyme (E sigmaF) containing sigmaF selectively transcribed the flagellar and chemotaxis genes, all of which could not be transcribed by E sigma70. This strict promoter recognition property of sigmaF is similar to those of other stress response minor sigma subunits but different from those of the principal sigma subunits, sigma70 and sigma38. sigma70-dependent transcription in vitro is inhibited at high concentrations of all salts tested, showing maximum activity at 50 mM. In contrast, sigmaF-dependent transcription was maximum at 50 mM KCI and then decreased to negligible level at 300 mM; in the cases of potassium acetate and potassium glutamate, maximum transcription was between 200 and 300 mM. DNase I foot printing of the fliC and fliD promoters indicated that sigmaF alone is unable to bind DNA, but E sigmaF specifically recognizes -10 and -35 regions of the sigmaF-dependent promoters with rather long upstream protection. Alteration of the promoter structure after binding of E sigmaF was suggested.