Pronounced changes in neuronal morphology occur as synapses mature; however, little is known about how synaptic transmission regulates the developing neuronal cytoskeleton. The postsynaptic, microtubule-associated protein MAP2 is a target of multiple, calcium-dependent signaling pathways activated by synaptic transmission. Here we demonstrate that MAP2 phosphorylation is differentially regulated across development. In 32P-labeled hippocampal slices prepared from adult rats, depolarization stimulated a bidirectional change in the phosphorylation of immunoprecipitated MAP2. A transient increase was mediated by metabotropic glutamate receptors (mGluRs) and stimulation of mitogen-activated protein kinases (MAPKs), Ca2+/calmodulin-dependent protein kinases (CaMKs), and protein kinase C (PKC). This increase was followed by a persistent dephosphorylation mediated by NMDA receptors and activation of protein phosphatase 2B (PP2B or calcineurin). In contrast, depolarization of neonatal hippocampal slices stimulated exclusively a net increase in MAP2 phosphorylation, which was attenuated by inhibitors of MAPKs, but not CaMKs or PKC. Furthermore, although incubation in NMDA induced a time-dependent decrease in MAP2 phosphorylation in both adults and neonates, this effect was both less robust and less sensitive to calcineurin inhibitors in neonates than in adults. These data indicate that the mechanisms coupling glutamate release to MAP2 dephosphorylation are relatively lacking in the neonatal hippocampus. Highly phosphorylated MAP2 is impaired in its ability to stabilize microtubules and actin filament bundles in vitro. The neonatal propensity toward glutamate-stimulated MAP2 phosphorylation may serve to reduce cytoskeletal stability and permit dendritic arborization early in postnatal development. In mature neurons, the bidirectional control of MAP2 phosphorylation may participate in activity-dependent synaptic remodeling.