RNA polymerase holoenzyme from Micrococcus luteus synthesizes in vitro a run-off transcript of 85 nucleotides from a DNA fragment containing part of gene E of bacteriophage phi X174. This RNA starts with GTP as the 5' terminus 18 nucleotides downstream from the start of gene E on the viral (+)strand. Transcription does not occur when the fragment is cleaved 36 nucleotides upstream of the initiation site. No transcript is obtained with RNA polymerase core or holoenzyme from Escherichia coli. Other DNA fragments containing the three major E. coli promoters of phi X174 are transcribed by both enzymes although much less efficiently by M. luteus RNA polymerase. When subunit sigma in E. coli RNA polymerase is replaced by sigma from M. luteus the resulting hybrid enzyme actively transcribes the DNA fragment containing the inner region of gene D with formation of the same run-off transcript which is obtained with M. luteus holoenzyme. In the presence of sigma from E. coli this RNA is not synthesized. The hybrid enzyme also transcribes a DNA fragment containing the gene A promoter of phi X174 with even higher efficiency than RNA polymerase holoenzyme from E. coli.