We have identified a cellular transcription factor from uninfected HeLa cells that stimulates the simian virus 40 (SV40) late mode of transcription and specifically binds the SV40 21-base-pair repeat promoter element. In particular, the late SV40 transcription factor (LSF) stimulates transcription at initiation sites L325 and L264 of the SV40 late promoter, which are the major transcription sites utilized after DNA replication during the SV40 lytic cycle. In addition, LSF appears to stimulate transcription to a lesser extent from the late-early initiation site of the early promoter. LSF binds specifically to the 21-base-pair repeats of the SV40 promoters, forming specific protein-DNA complexes, which migrate more rapidly through nondenaturing polyacrylamide gels than that formed by the previously identified transcription factor Sp1. Thus, LSF is distinguishable from Sp1 in both its transcriptional and DNA-binding properties. These findings suggest a potential role of LSF in the early to late transcriptional switch during a SV40 lytic infection.