We have characterized a transcription factor, obtained from simian virus 40 (SV40) chromosomes, which activates transcription from the SV40 late promoter in vitro. The late promoter-activating factor was distinct from SV40 T antigen as judged by its behavior on chromatography on hydroxylapatite; it was not recognized by anti-T antibodies, while T antigen itself was recognized. T antigen from SV40 chromosomes, on the other hand, abolished transcription in vitro from the early promoter. In DNase I footprinting experiments, a partially purified late promoter-activating factor preparation protected a region of DNA centered on SV40 nucleotide 270, which is between the repeated 72-base-pair enhancer and the major late RNA start site. Proteins from HeLa cells did not give the same footprint at this position. Gel mobility shift assays showed that proteins from SV40-infected CV-1 cells form a complex with DNA containing this binding site. The complex has a different rate of gel migration and a higher stability than complexes formed with proteins from uninfected cells.