After infection of Escherichia coli with bacteriophage T4 a series of modifications of RNA polymerase takes place including the association of several small polypeptides. We isolated RNA polymerase from cells abortively infected with a series of T4 mutants which arrest phage development at different stages and found that different sets of associated proteins are present in RNA polymerase in each case. The patterns of associated polypeptides seem to correlate with DNA content in the infected cells, suggesting that some of them can be involved both in DNA replication and in the transcription apparatus. One of the modified forms of RNA polymerase contains stoichiometric amounts of a protein with Mr = 25,000 (25K protein), which remains associated with the core enzyme after the removal of sigma factor by chromatography on phosphocellulose. The 25K protein was purified to homogeneity and its effect on transcription selectivity was analyzed in an in vitro system using fragments of T4 DNA as templates. The 25K protein exists in two functional forms which direct core RNA polymerase to utilize two different types of transcription start sites (class I and class II promoters). Both activities do not require host sigma factor. The two forms of 25K protein seem to compete with each other for the core enzyme. The isolated 25K protein can form stable dimers, suggesting that its two activities are associated with the dimeric and monomeric forms. Class I (but not class II) promoters can also be utilized in response to the host sigma factor.(ABSTRACT TRUNCATED AT 250 WORDS)