Two heat-stable protein factors, designated as H(1) and H(2), have been purified from a DNA-protamine sulfate complex obtained in an early step in the preparation of E. coli RNA polymerase. Gel electrophoresis under denaturing conditions indicates that H(1) and H(2) behave as pure entities, with molecular weights below 10,000. Their purity is confirmed by aminoacid composition data, and in the case of H(1), by immunological assays. H(1) and H(2) both strongly stimulate transcription of DNA from bacteriophages lambda and varphi80 by the E. coli polymerase holoenzyme; no effect was observed with single-stranded templates. That the amount of H(1) required for maximal stimulation is proportional to the amount of DNA present in the assay, and that both H(1) and H(2) strongly bind to native DNA in a synergestic fashion, suggests that these low molecular weight factors stimulate RNA synthesis by modifying the properties of the DNA template.