The core enzyme of protein phosphatase 2A is composed of a regulatory subunit A and a catalytic subunit C. It is controlled by three types of regulatory B subunits (B, B', and B") and by tumor (T) antigens, which are unrelated by sequence but bind to overlapping regions on the A subunit. To find out whether the different B subunits and T antigens bind to identical or distinct amino acids of the A subunit, mutants were generated and their abilities to bind B subunits and T antigens were tested. We found that some amino acids are involved in the binding of all types of B subunits, whereas others are specifically involved in the binding of one or two types of B subunits. T-antigen-binding specificity does not correlate with that of a particular type of B subunit.