THE NMDA (N-methyl D-aspartate) receptors in the brain play a critical role in synaptic plasticity, synaptogenesis and excitotoxicity^1–3. Molecular cloning has demonstrated that NMDA receptors consist of several homologous subunits (NMDAR1, 2A–2D)^4–7. A variety of studies have suggested that protein phosphorylation of NMDA receptors may regulate their function^7–12 and play a role in many forms of synaptic plasticity such as long-term potentiation^13,14. We have examined the phosphorylation of the NMDA receptor subunit NMDAR1 (NR1) by protein kinase C (PKC) in cells transiently expressing recombinant NR1 and in primary cultures of cortical neurons. PKC phosphorylation occurs on several distinct sites on the NR1 subunit. Most of these sites are contained within a single alternatively spliced exon in the C-terminal domain, which has previously been proposed to be on the extracellular side of the membrane^4,5,15. These results demonstrate that alternative splicing of the NR1 messenger RNA regulates its phosphorylation by PKC, and that mRNA splicing is a novel mechanism for regulating the sensitivity of glutamate receptors to protein phosphorylation. These results also provide evidence that the C-terminal domain of the NR1 protein is located intracellularly, suggesting that the proposed transmembrane topology model for glutamate receptors may be incorrect.