Protein phosphatase 2A (PP2A) is a major intracellular protein phosphatase that regulates multiple aspects of cell growth and metabolism. The ability of this widely distributed heterotrimeric enzyme to act on a diverse array of substrates is largely controlled by the nature of its regulatory B subunit. Only two gene families encoding endogenous B subunits have been cloned to date, although the existence of several additional regulatory subunits is likely. We have identified by two-hybrid interaction a new human gene family encoding PP2A B subunits. This family, denoted B56, contains three distinct genes, one of which is differentially spliced. B56 polypeptides co-immunoprecipitate with PP2A A and C subunits and with an okadaic acid-inhibitable, heparin-stimulated phosphatase activity. The three B56 family members are 70% identical to each other but share no obvious homology with previously identified B subunits. These phosphatase regulators are differentially expressed, with B56 alpha and B56 gamma highly expressed in heart and skeletal muscle and B56 beta highly expressed in brain. The identification of this novel phosphatase regulator gene family will facilitate future studies on the control of protein dephosphorylation and the role of PP2A in cellular function.