1. N-methyl-D-aspartate (NMDA) channels have two agonist binding sites that have similar binding rates for glutamate. However, it is not known whether the dissociation rates at these two sites, and hence their affinities, are similar. The competitive antagonist, D-2-amino-5-phosphonopentanoic acid (AP5), was used to study dissociation kinetics from NMDA receptors in outside-out patches from cultured hippocampal neurons. 2. Rapid steps from AP5 into NMDA produced currents with a sigmoidal activation time course that was limited by AP5 dissociation. Ensemble average currents were well fitted using kinetic models with two identical, cooperative antagonist binding sites per channel. The results suggest that the two NMDA binding sites have similar affinities, but that occupation of one site reduces the affinity of the other. 3. The agonist and antagonist binding kinetics are consistent with an approximately homogeneous population of NMDA channels in cultured hippocampal neurons.