We have used affinity chromatography on columns containing immobilized calf thymus RNA polymerase II to isolate three phosphoproteins (RAP72, RAP38, and RAP30) that bind directly to RNA polymerase II. All could be isolated from cell nuclei, and all three could be detected in mouse and human tissue culture cell lines, but only RAP38 and RAP30 have so far been isolated from calf thymus. RAP38 stimulates nonspecific transcription of native DNA templates by RNA polymerase II in the presence of Mn2+; it appears to be similar or identical to SII, a previously identified RNA polymerase II stimulatory factor (Nakanishi, Y., Mitsuhashi, Y., Sekimizu, K., Yokoi, H., Tanaka, Y., Horikoshi, M., and Natori, S. (1981) FEBS Lett. 130, 69-72). Unlike RAP38, RAP72 and RAP30 do not affect nonspecific transcription by RNA polymerase II. However, RAP30 may have a role in regulating some alterations of transcription that accompany cellular differentiation; RAP30 is partially dephosphorylated when murine erythroleukemia cells are induced with dimethyl sulfoxide to undergo terminal erythroid differentiation. We suggest that phosphate groups in RNA polymerase II-binding proteins may regulate transcription by modulating the interaction of RNA polymerase II with other regulatory proteins that possess sequence recognition specificity.