Adenovirus E4orf4 protein has been shown to induce p53-independent, protein phosphatase 2A (PP2A)-dependent apoptosis in transformed cells. Furthermore, E4orf4 also induces toxicity in Saccharomyces cerevisiae in a PP2A-dependent manner (D. Kornitzer and T. Kleinberger, submitted for publication). In this work, we utilized yeast cells to select for nonapoptotic E4orf4 mutants which, in turn, were shown to possess a diminished ability to bind PP2A. The success of this selection system will provide additional apoptosis-relevant mutants for E4orf4 research and strongly supports the relevance of E4orf4-induced toxicity in S. cerevisiae to E4orf4-induced apoptosis in mammalian cells.