The biphenyl derivative of 1,3-naphthalene disulfonic acid, known as Evans blue (EB), has been shown previously to specifically antagonize currents mediated by the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subtype of glutamate receptors (1). In contrast, we demonstrate herein that EB potently inhibits glutamate-evoked currents mediated by the kainate-type receptor GluR6 (IC50 150 nM) as well as the AMPA-type receptor GluR1 (IC50 = 220 nM) in whole-cell patch clamp recordings from transfected human embryonic kidney 293 cells. In addition to diminishing GluR6-mediated peak current amplitude, EB significantly altered receptor desensitization by slowing the rate of onset by approximately 2-fold (1 microM EB), slowing the rate of recovery by approximately 2-fold (0.1 microM EB), and increasing the steady state to peak current amplitude ratio by approximately 50-fold (1 microM EB). Interestingly, relatively little EB inhibition of GluR6 currents was observed in recordings from cells pretreated with the lectin concanavalin A, which eliminates kainate receptor desensitization. Similarly, currents recorded from GluR1-transfected cells were also relatively insensitive to EB inhibition if desensitization was first blocked by cyclothiazide. Moreover, for both GluR6 and GluR1, EB inhibition of agonist-evoked current was largely reversed if transfected cells were subsequently exposed to concanavalin A or cyclothiazide, respectively. Although EB may not be as selective an antagonist as previously believed, the relationship between EB-induced peak current inhibition and effects on receptor desensitization may be useful in further elucidating structures or mechanisms involved in the rapid desensitization of AMPA- and kainate-type glutamate receptors.