RNA polymerase requires one of a family of sigma subunits for specific promoter recognition and initiation. We have developed an in vitro method to define the RNA polymerase sigma subunit required by a promoter. Mouse monoclonal antibodies specific for either Escherichia coli sigma 70 or sigma 32 or Salmonella typhimurium sigma 54 were added to an E. coli coupled transcription-translation S-30 extract programed with a DNA template containing the promoter of interest. Using the representative lacUV5, glnAP2, and rpoDHS promoters as controls, we found that monoclonal antibodies to a given sigma subunit strongly inhibited transcription from cognate promoters which utilized that sigma subunit, but had little effect on transcription from noncognate promoters which used other sigma subunits. Supplementation of the S-30 extract with purified sigma 70, sigma 54, or RNA polymerase sigma 32-holoenzyme stimulated expression from the cognate promoters and inhibited noncognate promoters. These two tests, addition of monoclonal antibodies and addition of sigma subunits, provide a rapid means of identifying whether the sigma subunit required by any promoter expressed in the S-30 extract is sigma 70, sigma 54, or sigma 32. We suggest that this method may provide a systematic approach for identifying promoters which use as yet uncharacterized sigma subunits.