To investigate the properties of NMDA receptors expressed in new-born rat hippocampal granule cells, recordings were made of single-channel currents produced by application of glutamate or NMDA to outside-out membrane patches. Outside-out patches displayed two distinct patterns of single-channel activity. In some patches only high conductance single-channel activity composed of 42 and 50 pS currents was observed while in others both high (42 and 50 pS) and low (17 and 33 pS) conductance single-channel currents occurred. An absence of direct transitions connecting the smallest (17 pS) and largest (50 pS) conductance unitary currents, as well as an absence of direct transitions connecting 17, 42 and 50 pS currents in sequence, suggested that high and low conductance single-channel activity may have been produced as a result of the activation of two distinct NMDA receptor populations. The NR2B subunit-selective NMDA receptor antagonist, ifenprodil, blocked the high conductance currents suggesting that these receptors contain the NR2B subunit while a clear asymmetry in the frequency of direct transitions between 17 and 42 pS conductance levels indicates the presence of NMDA receptors containing NR2D subunits. In patches containing both high and low conductance-channel activity, evidence for negative coupling between NR2B- and NR2D-like channel activity was observed, suggesting receptors containing these subunits do not gate independently or that both NR2B and NR2D subunits may be part of a single receptor molecule. We conclude that NMDA receptors in P0 hippocampal granule cells are likely to be a mixture of NR1/NR2B diheteromers and receptors of novel molecular composition that may be triheteromeric receptors composed of NR1, NR2B and NR2D subunits.