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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 fluorescence calcium indicator fluo-3 AM. NR2B antagonists were added after stimulation of NMDA receptors with 10 microM glutamate and 10 microM 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 x 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.