Transcription complexes of simian virus 40 can be isolated from cells late in infection in a form that retains the ability to continue transcription in vitro. These complexes have been investigated previously to gain information about the nucleoprotein structure of a transcribing gene. However, several studies have reported that the RNA polymerase molecules in such complexes are located almost entirely at the 5' end of the transcription unit. This would indicate that these complexes cannot be those which were in the process of transcribing mRNA in the cell, because the transcribing complexes would be expected to contain polymerase that were distributed throughout the transcription unit at the time of extraction. Since this issue is important to the interpretation of studies which characterize the extracted complexes, we have determined the polymerase locations using two new approaches. The first employs a new application of a procedure in which the transcription complex DNA is radioactively tagged by transcription in vitro. The second is a new method, which analyzes run-off transcripts generated in vitro. Both methods demonstrate that the polymerases are distributed throughout the genome in the transcription complexes. This result indicates that these complexes were in the process of transcribing mRNA at the time of cell lysis.