Bacillus subtilis core RNA polymerase, containing a His(6)-fusion to the C-terminus of the beta' subunit, was isolated by Ni-NTA, Superdex 200 gel filtration, and Mono Q anion-exchange chromatography. The purified core enzyme was shown to be free of the major sigma factor sigma(A) and the transcription factors NusA and GreA. The purification procedure can be completed within 1 working day, is scalable, and yields highly purified and active core RNA polymerase.