NMDA receptor (NMDAR) subunits ε1–ε4 are expressed differentially with respect to brain region and ontogenic period, but their functional roles still are unclear. We have compared an ε1 subunit-ablated mutant mouse with the wild-type to characterize the effect of ε subunit expression on NMDAR-mediated single-channel currents and synaptic currents of granule cells in cerebellar slices. Single-channel and Western blot analyses indicated that the ε2 subunit disappeared gradually during the first postnatal month in both wild-type and mutant mice. Concomitantly, the voltage-dependent Mg2+block of NMDAR-mediated EPSCs (NMDA-EPSCs) was decreased. Throughout the developmental period studied, postnatal day 7–24 (P7–P24), the decay time course of NMDA-EPSCs in ε1 mutant (−/−) mice was slower than in wild-type mice. We suggest that the expression of the ε3 subunit late in development is responsible for a reduction in the sensitivity of NMDA-EPSCs to block by extracellular Mg2+and that receptors containing the ε1 subunit determine the fast kinetics of the NMDA-EPSCs.