The introduction of SV40 small t antigen or the suppression of PP2A B56; subunit expression contributes to the experimental transformation of human cells. To investigate the role of cancer-associated PP2A AA subunit mutants in transformation, we introduced several PP2A AA mutants into immortalized but nontumorigenic human cells. These PP2A AA mutants exhibited defects in binding to other PP2A subunits and impaired phosphatase activity. Although overexpression of these mutants failed to render immortalized cells tumorigenic, partial suppression of endogenous PP2A AA expression activated the AKT pathway and permitted cells to form tumors in immunodeficient mice. These findings suggest that cancerassociated AA mutations contribute to cancer development by inducing functional haploinsufficiency, disturbing PP2A holoenzyme composition, and altering the enzymatic activity of PP2A.(Cancer Res 2005; 65 (18): 8183-92)