Purified non-histone proteins from mouse mammary cells bind specifically to homologous DNA or chromatin. Complexes of non-histone protein with DNA or chromatin, isolated on agarose columns, were transcribed with both Escherichia coli RNA polymerase and RNA polymerase B from calf thymus. The fact that complexing of DNA with non-histone proteins increases transcription by E. coli RNA polymerase but not by RNA polymerase B suggests different mechanisms of transcription by these two enzymes. Similar experiments with mouse and Drosophila chromatin indicate that non-histone proteins specifically stimulate the transcription of mouse chromatin by RNA polymerase B. Non-histone proteins stimulate the transcription of mouse mammary tumor virus sequences in chromatin by RNA polymerase B but not by E. coli RNA polymerase. We conclude that those non-histone proteins bound specifically to chromatin are able to activate the transcription of specific genes by eukaryotic RNA polymerase.