The E2 open reading frame (ORF) of the bovine papillomavirus (BPV-1) encodes a family of site-specific DNA-binding proteins. The full-length protein is a transcriptional activator, whereas the polypeptides that contain only the carboxy-terminal domain are repressors. Here we show that the trans-activator can work as a repressor of transcription for one of the BPV-1 promoters by binding to a DNA sequence required for basal activity of the promoter. This operator site is defined as a 12-bp sequence that lies immediately downstream of the cap site. The operator DNA contains sequences that are defined genetically and biochemically as being important for basal level promoter activity. Furthermore, this site has been shown to be protected in a DNase footprint assay using fractionated HeLa cell extracts. The repression does not simply result from E2 blocking RNA polymerase initiation or elongation, because a strong E2-binding site placed at the operator has no repressive effect on transcription when the basal target sequence is placed independently upstream of the promoter. Thus, this is an interesting parallel to a theme well known in prokaryotes, where some site-specific DNA-binding proteins can work as either activators or repressors. In this system, as well as in the prokaryotic systems, the precise position of the binding site relative to other cis signals at the promoter determines the nature of the effects.