We studied the action potential-evoked autaptic N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) using solitary cerebellar neurons cultured in microislands from wild-type (+/+), NR2A subunit knockout (NR2A-/-) and NR2C subunit knockout (NR2C-/-) mice. The peak amplitude of autaptic NMDA-EPSCs increased for all genotypes between day in vitro 8 (DIV8) and DIV13. Compared to +/+ cells at DIV13, NR2A-/- cells had smaller and NR2C-/- cells larger NMDA-EPSCs. The decay time of these currents were all unexpectedly fast, except in NR2A-/- neurons, and showed small but significant shortening with development. Comparison of quantal parameters during development indicated an increase in quantal content in all genotypes. The synaptic portion of NMDA receptors measured using MK-801 blockade was approximately 50% in all genotypes at DIV8, and this percentage became slightly larger in NR2A-/- and NR2C-/- neurons at DIV12. The NR2B-selective antagonists ConantokinG and CP101,606 differed in their blocking actions with development, suggesting the presence of both heterodimeric NR1/NR2B and heterotrimeric NR1/NR2A/NR2B receptors. The most striking result we obtained was the significant increase of NMDA-EPSC peak amplitude and charge transfer in NR2C-/- mice. This was mainly due to an increase in quantal size as estimated from miniature NMDA-EPSCs. The expression of NR2C subunit containing receptors was supported by the decreased Mg²⁺ sensitivity of NMDA receptors at DIV13 in +/+ but not in NR2C-/- cells. Thus, solitary cerebellar granule neurons provide a novel model to investigate the role of receptor subtypes in the developmental changes of synaptic NMDA receptors.