Protein phosphatase 2A (PP2A) contains a 36-kDa catalytic subunit (PP2Ac), a 65-kDa structural subunit (PR65/A), and a regulatory B subunit. The core enzyme consists of the structural and catalytic subunits. The catalytic subunit exists as two closely related isoforms, alpha and beta. Several natural toxins, including okadaic acid (OA) and microcystins, specifically inhibit PP2A. To obtain biologically active recombinant PP2A and to compare the properties of the PP2A catalytic subunit alpha and beta isoforms, we expressed human PP2Ac alpha and c beta in High Five insect cells. The recombinant PP2Ac alpha, and c beta possess similar phosphatase activities using p-NPP and phosphopeptide as substrates and are strongly inhibited by OA and microcystin-LR to similar degrees. In addition, PP2Ac alpha or c beta was co-expressed with PR65/A and Co-purified as a core dimer, PP2A(D) (A alpha/c alpha and A alpha/c beta) with PR65 alpha/A alpha. The recombinant PP2A, bound to the B subunit in vitro. These results show that the recombinant PP2Ac alpha and c beta are identical in their ability to associate with the A and B subunits, in their phosphatase activities, and in carboxyl-methylation. Furthermore, our results show that High Five insect cells can produce biologically active recombinant PP2A, which should be a valuable tool for detecting natural toxins and investigating the mechanism of PP2A catalysis and other protein interactions. (C) 2005 Elsevier Inc. All rights reserved.