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A soluble extract prepared from human cells (KB S-100) has been recently shown to direct accurate transcription initiation by purified RNA polymerase II at the major late promoter of adenovirus 2. We have fractionated this extract by chromatography on phosphocellulose, DEAE-cellulose, and DNA-cellulose, and have identified four components that are required for the active and selective initiation of transcription by RNA polymerase II at this promoter. One of these components seems to act by suppressing random, but not selective, transcription by RNA polymerase II. All but one of these components have been shown to be chromatographically distinct from the factors involved in directing selective transcription by RNA polymerase III.