The adenovirus E4orf6 protein is shown here to interact with the cellular tumor suppressor protein p53 and to block p53-mediated transcriptional activation. The adenovirus protein inhibited the ability of p53 to bind to human TAF(II)31, a component of transcription factor IID (TFIID). Earlier work demonstrated that the interaction of p53 with TAF(II)31 involves a sequence near the NH2-terminus of p53, whereas the E4orf6-p53 interaction occurs within amino acids 318 to 360 of p53. Thus, the E4orf6 protein interacts at a site on p53 distinct from the domain that binds to TAF(II)31 but nevertheless inhibits the p53-TAF(II)31 interaction.