The A subunit of protein phosphatase 2A (PP2A) consists of 15 nonidentical repeats. The catalytic C subunit binds to C-terminal repeats 11 - 15 and regulatory B subunits bind to N-terminal repeats 1 - 10. Recently, four cancer-associated mutants of the A-alpha subunit have been described: Glu64-->Asp in lung carcinoma, Glu64-->Gly in breast carcinoma, Arg418-->Trp in melanoma, and Delta171 - 589 in breast carcinoma. Based on our model of PP2A, we predicted that Glu64-->Asp and Glu64-->Gly might be defective in B subunit binding, whereas Arg418-->Trp and Delta171 - 589 might bind neither B nor C subunits. We generated these mutants by site-directed mutagenesis and assayed their ability to associate with different forms of B subunits (B, B', B") or with the catalytic C subunit. The results demonstrate that all mutants are defective in binding either B or B and C subunits. Specifically, the N-terminal mutants, Glu64-->Asp and Glu64-->Gly, are defective in B' but normal in B, B", and C subunit binding, whereas the C-terminal mutants Arg418-->Trp and Delta171 - 589 bind none of the B subunits nor the C subunit. The implications of these findings with regard to the potential role of PP2A as a tumor suppressor are discussed. Oncogene (2001) 20, 10 - 15.