Protein phosphatase 2A (PP2A), a major phospho-serine/threonine phosphatase, is conserved throughout eukaryotes. It dephosphorylates a plethora of cellular proteins, including kinases and other signaling molecules involved in cell division, gene regulation, protein synthesis and cytoskeleton organization. PP2A enzymes typically exist as heterotrimers comprising catalytic C-, structural A- and regulatory B-type subunits. The B-type subunits function as targeting and substrate-specificity factors; hence, holoenzyme assembly with the appropriate B-type subunit is crucial for PP2A specificity and regulation. Recently, several biochemical and structural determinants have been described that affect PP2A holoenzyme assembly. Moreover, the effects of specific post-translational modifications of the C-terminal tail of the catalytic subunit indicate that a 'code' might regulate dynamic exchange of regulatory B-type subunits, thus affecting the specificity of PP2A.