The adenovirus E4orf4 protein when expressed alone at high levels induces the death of human cancer cells but not normal primary cells. It also is toxic in the yeast Saccharomyces cerevisiae, which we have used as a model system in some studies. Toxicity induced by the E4orf4 protein is largely dependent on its ability to associate with the highly conserved B/B55/Cdc55 class of regulatory subunits of protein phosphatase 2A (PP2A), of which the mammalian B55α species is best characterized structurally. We showed previously that binding to B55α appears to inhibit PP2A activity against at least some substrates. In the present study, we mapped the E4orf4 binding site on both yeast Cdc55 and mammalian B55α and propose how such binding may inhibit PP2A activity. The implications of E4orf4 binding on PP2A activity are of significant scientific interest in terms of the process by which PP2A recognizes and dephosphorylates its substrates. We also propose that E4orf4 binding in the context of viral replication serves the quite different function of introducing novel substrates for dephosphorylation by the PP2A holoenzyme.