The glutamate receptor channels of the N-methyl-D-aspartate (NMDA) subtype are composed of different subunits named NR1 and NR2A-D. These subunits can combine in different oligomers with diverging properties and their expression is developmentally regulated. We have used rat cerebellar slice cultures to test the involvement of bioelectrical activity and synaptic transmission in the changes in NR2A-C expression observed in developing granule cells. A correlation between the functional properties of the NMDA receptors and expression of the NR2A-C mRNAs was obtained in single granule cells by coupling patch-clamp recording and reverse transcription followed by polymerase chain reaction. Granule cells grown under standard culture conditions expressed mainly NR2A mRNA when examined after 15-40 days in vitro. Consistent with this observation, their responses to NMDA were only weakly reduced by 3 microM ifenprodil, a non-competitive antagonist which discriminates between NR2A and NR2B subunits in expression systems. In cerebellar cultures chronically exposed to tetrodotoxin to eliminate spontaneous electrical activity, granule cells maintained a predominant expression of NR2B subunits and their responses to NMDA were largely inhibited by 3 microM ifenprodil. These results provide evidence that the expression of the NR2A and B subunits is regulated through an activity-dependent mechanism leading to the formation of NMDA receptors with different pharmacological properties. Finally, the NR2C subunit, abundantly expressed in vivo by adult granule cells, was only rarely detected in slice cultures, even when excitatory synapses were formed between granule cells and fibres originating from co-cultured brainstem explants. These data suggest that the induction of NR2C expression observed in vivo requires an additional factor(s) that remains to be identified.