Compartmentalization of protein kinases and phosphatases with substrates is a means to increase the efficacy of signal transduction events. The A-kinase anchoring protein, AKAP79, is a multivalent anchoring protein that maintains the cAMP-dependent protein kinase, protein kinase C, and protein phosphatase-2B (PP2B/calcineurin) at the postsynaptic membrane of excitatory synapses where it is recruited into complexes with N-methyl-d-aspartic acid or alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)-subtype glutamate receptors. We have used cellular targeting of AKAP79 truncation and deletion mutants as an assay to map the PP2B-binding site on AKAP79. We demonstrate that residues 315-360 are necessary and sufficient for AKAP79-PP2B anchoring in cells. Multiple determinants contained within this region bind directly to the A subunit of PP2B and inhibit phosphatase activity. Peptides spanning the 315-360 region of AKAP79 can antagonize PP2B anchoring in vitro and targeting in transfected cells. Electrophysiological experiments further emphasize this point by demonstrating that a peptide encompassing residues 330-357 of AKAP79 attenuates PP2B-dependent down-regulation of GluR1 receptor currents when perfused into HEK293 cells. We propose that the structural features of this AKAP79-PP2B-binding domain may share similarities with other proteins that serve to coordinate PP2B localization and activity.