The calcineurin (CaN) a and b catalytic subunit isoforms are coexpressed within almost all cell types. The enzymatic properties of CaN heterodimers comprised of the regulatory B subunit (CnB) with either the a or b catalytic subunit were compared using in vitro phosphatase assays. CaN containing the a isoform (CnAa) has lower Km and higher Vmax values than CaN containing the b isoform (CnAb) toward the PO4RII, PO4-DARPP-32(20–38) peptides, and p-nitrophenylphosphate (pNPP).