Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme consisting of a catalytic subunit (C), a structural subunit (A), and a variable regulatory subunit (B). We have investigated the spatial and temporal expression patterns of three members of the B subunit family, Balpha, Bbeta, and Bgamma, both at the message level by using ribonuclease protection analysis and at the protein level by using specific antibodies. Although A, Balpha, and C protein are expressed in many tissues, Bbeta and Bgamma were detectable only in brain. Balpha, Bbeta, and Bgamma are components of the brain PP2A heterotrimer, because they copurified with A and C subunits on immobilized microcystin. Whereas Balpha and Bbeta are mainly cytosolic, Bgamma is enriched in the cytoskeletal fraction. In contrast to A, C, and Balpha, which are expressed at constant levels, Bbeta and Bgamma RNA and protein are developmentally regulated, with Bbeta levels decreasing and Bgamma levels increasing sharply after birth. RNA and immunoblot analyses of subdissected brain regions as well as immunohistochemistry demonstrated that B subunits are expressed in distinct but overlapping neuronal populations and cellular domains. These data indicate that B subunits confer tissue and cell specificity, subcellular localization, and developmental regulation to the PP2A holoenzyme. The Balpha-containing heterotrimer may be important in general neuronal functions that involve its partially nuclear localization. Holoenzymes containing B likely function in early brain development as well as in somata and processes of subsets of mature neurons. Bgamma may target PP2A to cytoskeletal substrates that are important in the establishment and maintenance of neuronal connections.