Multiple protein factors from HeLa cells are necessary for the accurate initiation of transcription on minimal promoters in vitro. We have partially purified these factors by chromatographic methods. In addition to RNA polymerase II, six factors A-F (FA, FB, FC, FD, FE, and FF) necessary for initiation at the beta-globin promoter start site in vitro have been identified. Certain of these (FA, FC, and FE) have been purified to near homogeneity. The present purification scheme yields sufficient amounts of purified material for the more detailed characterization and cloning of the genes for these activities. Among these factors, FD and FF were required with template DNA at an early step of formation of the initiation complex, whereas FB, FA together with FC, and FE were effective when added at successively later stages in the process of complex formation.