Phosphorylation of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluR1 at Ser(845) enhances AMPA channel activity. This study demonstrates that Ser(845) is rapidly dephosphorylated upon AMPA receptor activation in nucleus accumbens slices. AMPA-induced dephosphorylation at Ser(845) was blocked by CNQX, an AMPA receptor antagonist, by nifedipine, an L-type Ca(2+) channel antagonist, or by cyclosporin A, a calcineurin inhibitor. N-methyl-D-aspartate (NMDA) treatment also decreased phosphorylation of Ser(845), an effect that was blocked by MK-801, an NMDA receptor antagonist, but not by nifedipine. Accumbens neurons are enriched for dopamine- and cyclic AMP (cAMP)-regulated phosphoprotein, Mr 32,000 (DARPP-32), a potent inhibitor of protein phosphatase 1 (PP1) when phosphorylated by PKA (at Thr(34)). We tested the hypothesis that the AMPA/KA or NMDA-stimulated dephosphorylation of DARPP-32 via calcineurin, leading to increased PP1 activity and dephosphorylation of GluR1. AMPA or NMDA treatment decreased phospho-Thr(34)-DARPP-32 levels, effects that were blocked by receptor antagonists, or cyclosporin A. However, dephosphorylation of Ser(845) mediated by AMPA or NMDA receptors was unaffected in DARPP-32/inhibitor-1 knockout mice. These data suggest that AMPA- or NMDA-induced dephosphorylation of GluR1 at Ser(845) occurs by a mechanism that is independent of DARPP-32 and PP1, but involves activation of calcineurin. Thus, Ca(2+)-dependent dephosphorylation of GluR1 may serve as a negative feedback mechanism for the regulation of AMPA receptor activity in neurons.