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The NR1 subunit of the NMDA receptor has two serines (S890 and S896) whose phosphorylation by protein kinase C (PKC) differentially modulates NMDA receptor trafficking and clustering. It is not known which PKC isoforms phosphorylate these serines. In primary cultures of cerebellar neurons, we examined which PKC isoforms are responsible for the phosphorylation S890 and S896. We used specific inhibitors of PKC isoforms and antibodies recognizing specifically phosphorylated S890 or S896. The results show that PKC α phosphorylates preferentially S896 and PKC γ preferentially S890. Activation of type I metabotropic glutamate receptors (mGluRs) with DHPG (3,5-dihyidroxy-phenylglycine) activates PKC γ but not PKC α or β. We found that activation of mGluRs by DHPG increases S890 but not S896 phosphorylation, supporting a role for PKC γ in the physiological modulation of S890 phosphorylation. It is also shown that the pool of NR1 subunits present in the membrane surface contains phosphorylated S890 but not phosphorylated S896. This supports that differential phosphorylation of S890 and S896 by different PKC isoforms modulates cellular distribution of NMDA receptors and may also contribute to the selective modulation of NMDA receptor function and intracellular localization.