The Polyoma virus enhancer (A + B domain) activates transcription in Saccharomyces cerevisiae when joined to appropriate yeast promoter elements. We demonstrate by DNase I footprints and inhibition of binding by specific antibody, that the yeast protein RAP1 binds to the B-domain of the Polyoma enhancer and, at least in some promoter contexts, is responsible for transcriptional activity of the enhancer B-domain in yeast. Close matches to a consensus RAP1-binding site are also present in other viral enhancers.