The SV40 small t antigen (ST) interacts with the serine-threonine protein phosphatase 2A (PP2A). To investigate the role of this interaction in transformation, we suppressed the expression of the PP2A B56gamma subunit in human embryonic kidney (HEK) epithelial cells expressing SV40 large T antigen, hTERT, and H-RAS. Suppression of PP2A B56gamma expression inhibited PP2A-specific phosphatase activity similar to that achieved by ST and conferred the ability to grow in an anchorage-independent fashion and to form tumors. Overexpression of PP2A B56gamma3 in tumorigenic HEK cells expressing ST or human lung cancer cell lines partially reversed the tumorigenicity of these cells. These observations identify specific PP2A complexes involved in human cell transformation.