NMDA receptors regulate both the activation and inactivation of the extracellular signal-regulated kinase (ERK) signaling cascade, a key pathway involved in neuronal plasticity and survival. This bi-directional regulation of ERK activity by NMDA receptors has been attributed to opposing actions of NR2A- versus NR2B-containing NMDA receptors, but how this is implemented is not understood. Here, we show that glutamate-mediated intracellular Ca(2+) increases occur in two phases, a rapid initial increase followed by a delayed larger increase. Both phases of the Ca(2+) increase were blocked by MK-801, a non-selective NMDA receptor inhibitor. On the other hand, selective inhibition of NR2B-NMDA receptors by Ifenprodil or Ro 25-6981 blocked the delayed larger phase but had only a small effect on the rapid initial increase. The rapid initial increase in Ca(2+), presumably because of NR2A-NMDAR activation, was sufficient to activate ERK, whereas the large delayed increases in Ca(2+) mediated by NR2B-NMDARs were necessary for dephosphorylation and subsequent activation of striatal-enriched phosphatase, a neuron-specific tyrosine phosphatase that in turn mediates the dephosphorylation and inactivation of ERK. We conclude that the magnitude of Ca(2+) increases mediated through NR2B-NMDA receptors plays a critical role in the regulation of the serine/threonine and tyrosine kinases and phosphatases that are involved in the regulation of ERK activity.