Previous work with recombinant receptors has shown that the identity of the NMDA NR2 subunit influences receptor affinity for both glutamate and glycine. We have investigated the developmental change in NMDA receptor affinity for both glutamate and glycine in acutely dissociated parietal cortex neurons of the rat, together with the expression during ontogeny of NR2A and NR2B mRNA and protein. Whereas there is little change in NMDA receptor glutamate affinity with age, a population of NMDA receptors emerges in 14- and 28-d-old animals with a markedly reduced affinity for glycine (mKD = approximately 800 nM) and a reduced sensitivity to the NR2B subunit-selective NMDA antagonist ifenprodil. These changes are paralleled by a developmental increase in the expression of NR2A. Thus, in mature animals a population of NMDA receptors appears with a lower affinity for glycine that might not be saturated under normal physiological conditions. Ifenprodil (10 microM) inhibits virtually all of the NMDA receptor-evoked current in very young neurons that contain a single population of receptors exhibiting a high affinity for glycine (mKD = approximately 20 nM). In older neurons, which contain NMDA receptors with both high and low affinities for glycine, ifenprodil (10 microM) inhibits both the high-affinity population and a significant proportion of the low-affinity component, thus revealing three pharmacologically distinct populations of NMDA receptors in single neurons. Moreover, these observations suggest that ifenprodil might bind with high affinity to NMDA receptors containing both NR2A and NR2B subunits as well as those containing only NR2B.