Chromatin was isolated from SV40 transformed mouse cells (SV3T3) and transcribed with Escherichia coli RNA polymerase. The SV40 specific transcripts were analyzed by annealing the RNA to the minus strands of purified fragments of SV40 DNA produced by cleavage of the DNA with a restriction enzyme isolated from Hemophilus aegyptius. Quantitation of the frequency of transcription from the region (fragments A and D) was transcribed five to ten times more frequently than the remaining regions. These results are in good agreement with the transcription pattern observed in the transformed cell. In contrast, transcription of purified SV3T3 DNA by E. coli polymerase produced roughly equal frequencies of transcription from all regions of the integrated SV40 DNA. Comparison of the results with the known distribution of initiation sites for E. coli RNA polymerase on linear SV40 DNA indicates that the major initiation site is relatively unavailable in SV3T3 chromatin whereas other sites are available. This restriction is not observed when purified SV3T3 DNA is used as a template and must therefore result from the association of protein or other macromolecules with the DNA of the chromatin.