Accurate transcription by RNA polymerase II has previously been shown to require a HeLa cell fraction designated [AB] in addition to other components (Samuels, M., Fire, A., and Sharp, P. A. (1982) J. Biol. Chem. 257, 14419-14427). A factor which substituted for HeLa [AB] was identified in chromatographic fractions from calf thymus and was purified an estimated 9,000-fold or more. The final calf thymus [AB] fractions contained three polypeptide species with molecular weights of 19,600, 19,100, and 12,800, whose appearance correlated with the transcription factor enzymatic activity. The intact factor had a molecular weight of 25,600-35,000 based on gel filtration and sedimentation analysis and could be inactivated by treatment with high temperatures or with N-ethyl-maleimide. [AB] did not stimulate nonspecific nucleotide incorporation by pure RNA polymerase II, but it did interact with factor [DB] and promoter template DNA to form a functional intermediate preceding accurate initiation. The calf thymus factor thus was homologous to HeLa [AB] by both physical and functional criteria. In contrast to a previous suggestion that this factor has properties associated with actin the highly purified active fractions did not contain detectable actin.