Yeast cells contain an RNA polymerase factor, π, which is a heat-stable protein with an apparent molecular weight of 12,000. This factor stimulates transcription of calf-thymus, salmon-sperm, yeast-nuclear, and T4-phage DNA. It stimulates transcription by each of the four yeast-nuclear RNA polymerases, by rat-liver RNA polymerases I and II, and by Escherichia coli RNA polymerase. π-Factor can cause each of the eukaryotic RNA polymerases to become insensitive to rifamycin AF-013, but does not stop inhibition of E. coli RNA polymerase by rifamycin AF-013. Stimulation of transcription by π-factor is general, and does not apply only to a limited class of genes. Apparently, π-factor stimulates transcription by increasing the proportion of RNA polymerase binding events that leads to the initiation of RNA chains.