The transcription of chromatin from adenovrius 2 transformed rat cells by murine plasmacytoma RNA polymerases I, II and III has been studied. Both the total RNA synthesis and transcription of the integrated adenovirus 2 genes by RNA polymerase II represent de novo DNA transcription as assessed by their sensitivity to actinomycin D. It is shown that each RNA polymerase class has characteristic ionic strength activation profiles and metal ion requirements. RNA polymerase II transcribes the integrated adenovirus 2 genes in chromatin at a frequency 25- to 50-fold higher than their sequences are represented in the genome. In contrast, no detectable viral RNA is synthesized when deproteinized DNA is transcribed. In the presence of Mn2+, all three RNA polymerases (I, II and III) transcribe the integrated viral genes at approximately the same relative frequencey. However, the Mg2+ as divalent cation, the proportion of the total RNA which represents viral gene transcripts is increased 3- to 4-fold with RNA polymerase II, while it remains unchanged for RNA polymerases I or III.