Several viral transcriptional activators have been shown to interact with the basal transcription factor TATA-binding protein (TBP). These associations have been implicated in facilitating the assembly of the transcriptional preinitiation complex. We report here that the hepatitis B virus protein X (pX) specifically binds to TBP in vitro. While truncations of the highly conserved carboxyl terminus of TBP abolished this binding, amino-terminal deletions had no effect. Deletion analysis suggests that a domain consisting of 71 aa in the highly conserved carboxyl-terminal region of TBP is necessary for its interaction with pX. The minimal region in pX sufficient for its interaction with TBP includes aa 110-143. Furthermore, TBP from phylogenetically distinct species including Arabidopsis thaliana, Saccharomyces cerevisiae, Drosophila melanogaster, and Solanum tuberosum (potato) bound to pX. The pX-TBP interaction was inhibited in the presence of nonhydrolyzable analogs of ATP, suggesting a requirement for ATP. These results provide an explanation for the promiscuous behavior of pX in the transactivation of a large repertoire of cellular promoters. This study further implicates a fundamental role for pX in modulating transcriptional regulatory pathways by interacting with the basal transcription factor TBP.