Protein factors which stimulate R N A polymerase I1 but not polymerases I and 111 on native D N A have been isolated from calf thymus. These factors are basic proteins and exhibit properties which are similar to the stimulatory factor first described by H. Stein & P. Hausen [(1971) Cold Spring Harbor Symp. Quant. Biol. 35, 7 191. The stimulatory factors are initially purified by the following steps: homogenization, 40-80% ammonium sulfate precipitation, DEAE-Sephadex, CM-cellulose, and Sephadex G-75. Two stimulatory peaks (SF-1 and SF-2) are resolved by Sephadex G-75. SF-1 is resistant while SF-2 is sensitive to heat denaturation at 98 'C. The protein kinase activity present in SF-1 is heat labile, demonstrating that it is not responsible for the stimulation by SF-1. Both factors stimulate transcription on native calf thymus DNA but not denatured calf thymus DNA. SF-2 stimulates while SF-I does not stimulate transcription on poly[d(A-T)]. Both factors are sensitive to digestion with proteolytic enzymes, indicating that both factors contain a protein which is essential for stimulatory activity. Gel filtration studies show that SF-1 and SF-2 exhibit native molecular weights of D i f f e r e n t classes of eukaryotic RNA polymerase (Roeder and Rutter, 1969) transcribe the respective classes of cellular RNA. R N A polymerase