Simian Virus 40 (SV40) chromosomes were incubated with a concentrated extract of HeLa cells containing RNA polymerase II and other factors involved in transcription. SV40-specific RNA was synthesized. In the absence of HeLa cell extract the synthesis of labeled RNA by endogenous RNA polymerase in the chromosome preparations amounted to less than one tenth of that when the HeLa cell extract was present. Incubation with the HeLa extract increased the amount of Sarkosyl-resistant (i.e., transcribing) RNA polymerase on the SV40 chromosomes. When restriction endonucleases able to cut SV40 DNA were added to transcription reactions containing SV40 chromosomes and the HeLa cell extract, RNAs of discrete length were produced. The RNAs were identified as correctly initiated run-off transcripts of the early and late genes. Most of the RNA synthesized by the HeLa extract from SV40 chromosomes was from the region of the late genes, whereas transcription of purified SV40 DNA was from both the early and the late regions, with the early transcripts predominating.