Autophosphorylation of Ca2/calmodulin-dependent protein kinase II (CaMKII) at Thr286 generates Ca2~-independent activity. As an initial step toward understanding CaMKII inactivation, protein phosphatase classes (PP1, PP2A, PP2B, or PP2C) responsible for dephosphorylation of Thr286 in rat forebrain subcellular fractions were identified using phosphatase inhibitors/activators, by fractionation using ion exchange chromatography and by immunoblotting. PP2A-like enzymes account for >70% of activity toward exogenous soluble Thr286-autophosphorylated CaMKIl in crude cytosol, membrane, and cytoskeletal extracts; PP1 and PP2C account for the remaining activity. CaMKII is present in particulate fractions, specifically associated with postsynaptic densities (PSD5); each protein phosphatase is also present in isolated PSD5, but only PP1 is enriched during PSO isolation. When isolated PSDs dephosphorylated exogenous soluble Thr286-autophosphorylated CaMKll, PP2A again made the major contribution. However, CaMKII endogenous to PSDs (32P autophosphorylated in the presence of Ca 2+/calmodu tin) was predominantly dephosphorylated by PP1. In addition, dephosphorylation of soluble and PSD-associated CaMKII in whole forebrain extracts was catalyzed predominantly by PP2A and PP1, respectively. Thus, soluble and PSD-associated forms of CaMKII appear to be dephosphorylated by distinct enzymes, suggesting that Ca2-independent activity of CaMKII is differentially regulated by protein phosphatases in distinct subcellular compartments. Key Words: Protein kinaseProtein phosphatase-Calmodulin -Postsynaptic density-Synaptic plasticity.