The differential distribution of NMDA receptor subunit mRNAs in the developing mammalian cerebellum has been previously described. In this study, we investigated the temporal expression of NMDA receptor proteins in the postnatal murine cerebellum using antibodies specific for the NMDA zeta 1 and NMDA epsilon subunits. Our results showed a gradual increase during the first three weeks of life in the relative amount of NMDA zeta 1 and NMDA epsilon 1 proteins. In contrast, NMDA epsilon 2 increased transiently during this period, reaching a maximum around postnatal day 9 and decreasing thereafter to nearly undetectable levels by the end of the third week of life. The level of NMDA epsilon 3 increased dramatically between postnatal days 9 and 15 and thereafter remained constant. Immunoprecipitation of native proteins revealed that a large fraction of NMDA epsilon 2 was associated with NMDA zeta 1 and epsilon 1. At later developmental stages, NMDA epsilon 3 was predominantly assembled with NMDA zeta 1 but not with NMDA epsilon 1 or NMDA epsilon 2. These results demonstrate that NMDA receptor subtypes, formed by the assembly of different NMDA epsilon subunits with NMDA zeta 1, are sequentially expressed in the developing mouse cerebellum. The time course of their expression suggest, that these NMDA receptor subtypes may contribute to specific aspects of granule cell differentiation in the cerebellum.