Abstract: We have further analyzed the requirements of yeast RNA polymerase I (pol I) to initiate transcription at the ribosomal gene promoter. Resolution of yeast whole cell extracts through several chromatographic steps yielded three protein fractions required for accurate initiation. One fraction is composed of TBP associated within a 240 kDa protein complex. The fraction contributing the RNA polymerase I (pol I) activity consists of dimeric and monomeric pol I under conditions optimal for in vitro transcription. The capability to utilize the ribosomal gene promoter correlates with monomeric pol I complexes which are possibly associated with further transcription factors. These initiation competent pol I complexes appeared to be resistant to high salt concentrations. Pol I dimers which represent the majority of the isolated pol I, can be reversibly dissociated into monomers and are only active in non-specific RNA synthesis, if single stranded DNA serves as a template. We suggest a model in which dimeric inactive pol I is converted into an active monomeric form that might be associated with other transcription factors to maintain a stable initiation competent complex.