Abstract To facilitate the discovery of novel N-methyl- d-aspartate (NMDA) receptor antagonists, we have developed a high-throughput functional assay based on fluorescence detection of free intracellular calcium concentrations. Mouse fibroblast L(tk-) cells expressing human NR1a/NR2B NMDA receptors were plated in 96-well plates and loaded with florescence calcium indicator fluo-3 AM. NR2B antagonists were added after stimulation of NMDA receptors with 10 μM glutamate and 10 μM glycine. Changes in fluorescence after the addition of the antagonists were fitted by a single exponential equation providing k obs. The concentration dependence of k obs was linear for all NR2B antagonists at concentrations where k obs<0.2 s −1. The values of k obs for six structurally distinct NR2B antagonists were in the range of 1.1 to 7.5×10 5 M −1 s −1. These values were several orders of magnitude slower than that obtained for diffusion limited Mg 2+ channel block. The rate constants k off provided the values of t 1/2 for dissociation of NR2B antagonists in the range of 1.8 min for ifenprodil to 240 min for the slowest novel antagonist. The IC 50 values obtained from the end-point fluorescence measurements agree with K d values calculated from kinetic measurements. All kinetic constants, obtained using our fluorescence method, correlate well with data measured by voltage clamp.