Adenovirus E4orf4 protein is a multifunctional viral regulator, which is involved in down regulation of virally-modulated signal transduction, in control of alternative splicing of viral mRNAs, and in induction of apoptosis in transformed cells. It has been previously shown that E4orf4 interacts with protein phosphatase 2A through the phosphatase Balpha subunit. It was further shown that PP2A is required for performing the various E4orf4 functions. We report here that E4orf4 interacts with multiple isoforms of the PP2A-B' subunit, as well as with Balpha. We map the interaction sites of the B subunits on E4orf4 and show that they overlap but are not identical. We identify a dominant negative E4orf4 mutant, which disrupts the PP2A holoenzyme. We show that induction of apoptosis by E4orf4, which we previously reported to require the interaction with Balpha, is not affected by the interaction with B'. Our results suggest that the interaction of E4orf4 with various PP2A subpopulations may mediate the different E4orf4 functions.