Understanding the interaction between dopamine and glutamate, particularly the interaction of dopamine and NMDA receptors, may enable a more rational approach to the treatment of schizophrenia, drug addiction, and other psychiatric disorders. We show that, in prefrontal cortical neurons, dopamine D(1)-induced enhancement of NMDA receptor function depends on rapid insertion of new NMDA receptor 2B subunits on the synaptic surface. Protein kinase A (PKA) inhibitor, but not protein kinase C (PKC) inhibitor, completely blocked dopamine D(1) agonist SKF-81297-induced increase of the total expression of NMDA receptors. Furthermore, SKF-81297 failed to alter the surface expression and synaptic insertion of NMDA receptors in the presence of PKA inhibitor, phospholipase C inhibitor, PKC inhibitor, or Src family kinase inhibitor. Our data suggest that D(1)-mediated enhancement of NMDA current depends on the NMDA receptor trafficking through rapid synaptic insertion and both PKA and PKC signaling pathways play important roles in the regulatory process. Although both PKA and PKC mediate the D(1)-induced enhancement of NMDA receptors, the phospholipase C-PKC-Src pathway is only required for surface expression and new synaptic insertion of NMDA receptors.