Antagonism of glutamate-receptor responses activated by N-methyl-D-aspartic acid (NMDA) was studied using whole cell voltage clamp recording from mouse dissociated hippocampal neurons cultured for 10-15 d. The kinetics of onset of and recovery from NMDA receptor block during continuous application of NMDA together with either glycine, or L-alanine, were recorded in response to concentration jump application of NMDA- and glycine-binding site directed competitive antagonists, applied with a multibarrel flow pipe under conditions which allowed rapid solution changes around the cell less than 10 ms. Mathematical solutions for both one- and two-equivalent site models for competitive antagonism were determined according to the differential equations outlined by Colquhoun and Hawkes (1977. Proc. R. Soc. Lond. B. 199:231-262). The kinetics of action of D-CPP and D-AP5, NMDA binding site antagonists, and 7Cl-kynurenic acid, a glycine binding site antagonist, were examined for each model. For all these antagonists, the kinetics for the onset of and recovery from antagonism were better fit by the two-equivalent site model, which yielded antagonist microscopic kBoff/kBon values which closely approximated Ki values determined from analysis of equilibrium dose response curves. These results suggest that two molecules of NMDA and two molecules of glycine must bind to the NMDA receptor for activation of ion channel gating.