In rat cerebellar granule neurons, mRNA and protein levels of the NR2A and NR2C subunits of the NMDA receptor increase during the second postnatal week. At this time, mRNA and protein levels of the NR2B subunit begin to fall. To investigate targeting of NMDA receptor subunits, we performed whole-cell recordings from rat cerebellar granule neurons at different times during development and investigated the pharmacological and biophysical properties of mossy fiber-evoked NMDA EPSCs. Isolated NMDA EPSCs from newly formed synapses in the first postnatal week exhibited partial block by the NR2B subunit-specific antagonist (1S, 2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP 101,606). By the end of the second postnatal week, NMDA EPSCs were virtually unaffected by the NR2B antagonist. In parallel, NMDA EPSC decay times decreased over a similar developmental time course. We compared properties of synaptic NMDA receptors with extrasynaptic receptors that are present on the cell body with rapid application of glutamate to excised nucleated patches. Deactivation of patch responses accelerated with development and closely resembled evoked NMDA EPSCs in rats of the same age. However, patch responses were highly sensitive to CP 101,606 through the second postnatal week, and sensitivity was seen in some neurons up to the fourth postnatal week. Spermine potentiated peak NMDA patch responses from postnatal days 10-14 rats but had little effect on evoked NMDA EPSCs. Our data suggest selective targeting of a distinct NMDA receptor subtype to synaptic receptor populations in cerebellar granule neurons. Later in development, similar changes occur in the extrasynaptic receptor population.