Accumulating preclinical data suggest that compounds that block the excitatory effect of glutamate on the kainate subtype of glutamate receptors may have utility for the treatment of pain, migraine, and epilepsy. In the present study, the in vitro pharmacological properties of the novel glutamate antagonist 5-carboxyl-2,4-di-benzamido-benzoic acid (NS3763) are described. In functional assays in human embryonic kidney (HEK)293 cells expressing homomeric GLU(K5) or GLU(K6) receptors, NS3763 is shown to display selectivity for inhibition of domoate-induced increase in intracellular calcium mediated through the GLU(K5) subtype (IC(50) = 1.6 microM) of kainate receptors compared with the GLU(K6) subtype (IC(50) > 30 microM). NS3763 inhibits the GLU(K5)-mediated response in a noncompetitive manner and does not inhibit [(3)H]alpha-amino-3-hydroxy-5-tertbutylisoxazole-4-propionic acid binding to GLU(K5) receptors. Furthermore, NS3763 selectively inhibits l-glutamate- and domoate-evoked currents through GLU(K5) receptors in HEK293 cells and does not significantly inhibit alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid- or N-methyl-d-aspartate-induced currents in cultured mouse cortical neurons at 30 microM. This is the first report on a selective and noncompetitive GLU(K5) antagonist.