The functional interaction of thiocyanate (SCN-) ions with recombinant non-N-methyl-D-aspartate receptors was examined by studying α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and kainic acid (KA)-activated currents in Xenopus laevis oocytes. Recombinant receptors were expressed after microinjection of oocytes with combinations of cDNAs or cRNAs encoding for GluR1, GluR2, GluR2(R586Q), GluR3 or GluR6 subunits. When the GluR2 subunit was expressed with GluR1, SCN- (2 mM) inhibited the responses to 50 μM AMPA, whereas responses to 100 μM KA were slightly increased and responses to 200 μM L-glutamate were unaffected. Equilibrium concentration-response curves for AMPA were antagonized in a non-competitive manner by SCN- with a reduction in the EC50. The inhibitory effects of SCN- were unaffected by prior reduction of receptor desensitization with either 10 μM Concanavalin-A or 0.5 mM diazoxide. AMPA-activated currents recorded from homomeric GluR1 or GluR3 receptors were not affected by SCN-, and GluR6 homomeric receptors, which are sensitive to KA but not to AMPA, were also unaffected. In contrast, AMPA activation of homomeric GluR2(R586Q) subunit receptors, or combinations of GluR1 or GluR3 + GluR2(R586Q) subunits, were markedly inhibited by SCN-. In addition, the inhibitory effect of AMPA on KA-activated responses on these heteromeric receptors, was enhanced by SCN-. These results indicate that SCN- exert an inhibitory effect on ‘AMPA receptors’but only when the recombinant non-NMDA receptor is a GluR2 homomer, or when GluR2 subunits are present as part of a heteromeric combination. Moreover, this inhibitory effect was unaffected by the ‘Q/R’site in the presumed second transmembrane domain, since currents mediated by the GluR2(R586Q) subunit were also susceptible to inhibition by SCN-. Thus the inhibition was not related to the rectification properties or calcium permeability of the non-NMDA receptors. It is suggested that the GluR2 subunit may have a specific binding site for anions which could modulate the function of non-NMDA receptors and that SCN- may be a useful probe for the detection of these subunits in native neurons.