A series of gal promoter mutants has been used to compare the in vitro selectivities of the two forms of Escherichia coli RNA polymerase, E sigma 38 and E sigma 70. In the absence of the CRP-cAMP complex, E sigma 38 shows a strong preference for the ga/P1 promoter, whereas E sigma 70 preferentially initiates transcription from the ga/P2 promoter. E sigma 38 selectivity is not affected by the nature and position of the upstream sequences or by the phasing between synthetic upstream curved sequences and the -10 regions. In fact, all effects of mutations in the extended -10 region can be accounted for without evoking strong new sequence preferences for E sigma 38. Finally, both E sigma 38 and E sigma 70 initiate transcription from the ga/P1 promoter in the presence of CRP-cAMP complex and support direct cAMP-CRP activation at several CRP-dependent promoters.