The promoter selectivity of two extracytoplasmic function (ECF) subfamily sigma subunits, sigma(E) (sigma(24)) and sigma(FecI) (sigma(18)), of Escherichia coli RNA polymerase was analyzed by using an in vitro transcription system and various promoters. The Esigma(E) holoenzyme recognized only the known cognate promoters, rpoEP2, rpoHP3, and degP, and the Esigma(FecI) recognized only one known cognate promoter, fecA. The strict promoter recognition properties of sigma(E) and sigma(FecI) are similar to those of other minor sigma subunits. Transcription by Esigma(E) and Esigma(FecI) was enhanced by high concentrations of glutamate, as in the case of other minor sigma subunits. The optimum temperature for transcription by Esigma(FecI) was low, around 25 degrees C, apparently in agreement with the high rate of iron sequestration by E. coli at low temperatures. By quantitative Western blot analysis, the intracellular levels of sigma(E) and sigma(FecI) in the uninduced steady-state culture of E. coli W3110 (type A) were determined to be 0.7 to 2.0 and 0.1 to 0.2 fmol per microg of total proteins (or 3 to 9 and 0.4 to 0.9 molecules per cell), respectively, and less than 1% of the level of the major sigma(70) subunit.