The promoters of Micrococcus luteus, a bacterium whose chromosomal DNA has a high G + C content (74%), diverge from the consensus prokaryotic promoter in having GC-rich DNA sequences at less important positions (Nakayama, M., Fujita, N., Ohama, T., Osawa, S., and Ishihama, A. (1989) Mol. Gen. Genet. 218, 384-389). In order to compare the promoter selectivity of RNA polymerase between M. luteus and Escherichia coli, we purified the enzyme from both organisms. The sets of promoters recognized by the two RNA polymerases were found to overlap partly. Some, but not all, E. coli promoters were found to be correctly transcribed in vitro by M. luteus RNA polymerase as well as the E. coli enzyme. One molecular species of M. luteus sigma factor, with the apparent molecular mass of 60 kDa, was isolated from purified RNA polymerase. By the addition of either M. luteus or E. coli core enzyme it was reconstituted into active holoenzyme. Likewise, M. luteus core enzyme was reconstituted into a hybrid holoenzyme by the addition of E. coli sigma subunit. Both hybrid holoenzymes were, however, able to initiate transcription only from promoters which were recognized by both of the native holoenzymes.