Abstract Fractionation of HeLa cell extracts reveals the presence of a promoter-specific transcription factor, Sp1, which activates a class of promoters that includes the SV40 early promoter but not several others that have been tested. We analyzed SV40 early-promoter deletion mutants and determined that transcriptional activation by Sp1 requires sequences within tandem 21 bp repeats located 70–110 bp upstream of the transcription initiation sites. In a DNAase footprinting assay, Sp1 protected sequences in this same 21 bp repeat region, thus indicating the presence of a specific site for Sp1 binding. During purification of Sp1, there was a correlation between transcriptionstimulatory activity and promoter-binding activity. These results suggest that direct binding of Sp1 to sequences in the upstream promoter element is the mechanism by which this factor activates transcription by RNA polymerase II at the SV40 early promoter.