Evidence for post-recruitment functions of yeast transcription factor (TF)IIIB in initiation of transcription was first provided by the properties of TFIIIB-RNA polymerase III-promoter complexes assembled with deletion mutants of its Brf and B" subunits that are transcriptionally inactive because they fail to open the promoter. The experiments presented here show that these defects can be repaired by unpairing short (3 or 5 bp) DNA segments spanning the transcription bubble of the open promoter complex. Analysis of this suppression phenomenon indicates that TFIIIB participates in two steps of promoter opening by RNA polymerase III that are comparable to the successive steps of promoter opening by bacterial RNA polymerase holoenzyme. B" deletions between amino acids 355 and 421 interfere with the initiating step of DNA strand separation at the upstream end of the transcription bubble. Removing an N-terminal domain of Brf interferes with downstream propagation of the transcription bubble to and beyond the transcriptional start site.