N-methyl-D-aspartate receptor (NMDAR) stimulation activates many downstream mechanisms involved in both cell survival and cell death. The manner in which the NMDAR regulates one of these pathways, the p38 mitogen-activated protein kinase (p38) pathway, is currently unknown. In the present study, we have defined a developmental-, concentration-, and time-dependent phosphorylation and subsequent dephosphorylation of p38. In cultured hippocampal neurons 7-8 days in vitro (DIV7-8), NMDAR stimulation leads to a concentrationdependent increase in p38 phosphorylation (phosphop38). However, in more mature neurons (>DIV17) application of NMDA produces concentration-dependent effects, such that low concentrations result in sustained increases in phospho-p38 levels, and high concentrations dephosphorylate p38 within 5 min. Conantokin G, an antagonist of NR1/2A/2B and NR1/2B receptors, inhibits p38 phosphorylation, while NR1/2B-specific antagonists prevent the rapid dephosphorylation of p38 without affecting p38 activation. Furthermore, inhibition of calcineurin prevents the activation of p38, whereas inhibition of phosphoinositide 3-kinase (PI3K) prevents the rapid dephosphorylation of p38. Our results support the presence of subtype-dependent pathways regulating p38 activation and deactivation: one involves NR1/2A/2B receptors activating calcineurin and resulting in p38 phosphorylation, and the other utilizes NR1/2B receptors binding to and activating PI3K and leading to the dephosphorylation of p38 in a manner involving both NR1/2A/2B receptor activation and tyrosine phosphorylation of NR2B. The ability of NMDAR subtype-specific mechanisms to regulate p38 has implications for NMDAR-mediated synaptic plasticity, gene regulation, and excitotoxicity.The N-methyl-D-aspartate receptor (NMDAR), 1 a member of the ionotropic glutamate receptor family, is required for forms of neuronal synaptic plasticity, for cell death mediated by excitotoxicity and for gene regulation (1, 2). Differential responses mediated through NMDAR stimulation can be caused by the level of activation, the localization of the effect, or by the properties of the receptor. NMDARs likely contain two NR1 and two NR2 subunits (3). Most variations in receptor properties reflect differences in receptor subtype composition involving the four NR2 subunits (NR2A-NR2D) (2).In the hippocampus, NMDARs are composed mainly of NR1 subunits in combination with NR2A and NR2B subunits. The expression and localization of these NR2 subunits are developmentally regulated. Prenatally and early in postnatal development, hippocampal NMDARs are mostly NR1/2B receptors, localized at developing synapses with the synaptic-associated protein SAP-102 (4 -6). During maturation, expression of NR2A subunits increases, and NR2A-containing receptors, bound to post-synaptic density (PSD)-95, predominate in the synapse, whereas NR1/2B receptors become mostly extrasynaptic with SAP102 levels decreasing at the synapse (6 -10). NMDAR composition changes similarly in primary culture, in which imm...