In the developing cerebellum, switching of the subunit composition of NMDA receptors occurs in granule cells from NR2B-containing receptors to NR2C-containing ones. We investigated the mechanisms underlying switching of NR2B and NR2C subunit composition in primary cultures of mouse granule cells at the physiological KCl concentration (5 mM). Granule cells extensively extended their neuritic processes 48 h after having been cultured in serum-free medium containing 5 mM KCl. Consistent with this morphological change, NR2B mRNA and NR2C mRNA were down- and up-regulated, respectively, in the granule cells. This dual regulation of the two mRNAs was abrogated by blocking excitation of granule cells with TTX. This neuronal activity–dependent regulation of NR2B and NR2C mRNAs was abolished by the addition of selective antagonists of AMPA receptors and NMDA receptors. Furthermore, the dual regulation of NR2B and NR2C mRNAs in TTX-treated cells was restored by the addition of NMDA in the presence of the AMPA receptor antagonist, but not by that of AMPA in the presence of the NMDA receptor antagonist. Importantly, the NMDA receptor activation drove the NR2B/NR2C switching of NMDA receptors in the cell-surface membrane of granule cells. This investigation demonstrates that stimulation of NMDA receptors in conjunction with the AMPA receptor–mediated excitation of granule cells plays a key role in functional subunit switching of NMDA receptors in maturing granule cells at the physiological KCl concentration.