Protein kinases modulate the activity of several ligand-gated ion channels, including the NMDA (N-methyl-D-aspartate) subtype of glutamate receptor. Although phosphorylation and dephosphorylation of glutamate receptors may participate in several lasting physiological and pathological alterations of neuronal excitability, the physiological control of this cycle for NMDA channels has not yet been established. Using cell-attached recordings in acutely dissociated adult rat dentate gyrus granule cells, we now demonstrate that inhibitors of an endogenous serine/threonine phosphatase prolong the duration of single NMDA channel openings, bursts, clusters and superclusters. Okadaic acid, a non-selective phosphatase inhibitor, prolongs channel openings only at a concentration that inhibits the Ca2+/calmodulin-dependent phosphatase 2B (calcineurin), and is ineffective when Ca2+ entry through NMDA channels is prevented. Furthermore, FK506, an inhibitor of calcineurin, mimics the effects of okadaic acid. Thus in adult neurons, calcineurin, activated by calcium entry through native NMDA channels, shortens the duration of channel openings. Simulated synaptic currents were enhanced after phosphatase inhibition, which is consistent with the importance of phosphorylation of the NMDA-receptor complex in the short- and long-term control of neuronal excitability.