Joro spider toxin (JSTX) is one of the most potent antagonists of glutamatergic AMPA/KA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate) receptor channels in invertebrates and vertebrates. A differential blocking effect on certain types of glutamatergic synapses--e.g., parallel and climbing fiber synaptic inputs to rat cerebellar Purkinje neurons--has been shown by using a synthetic analog of the spider toxin. By investigating the molecular basis of the JSTX action on the recombinant AMPA/KA receptors GluR1-GluR4 and GluR6 expressed in Xenopus oocytes, we found that submicromolar concentrations of JSTX exert a subunit-specific block. Thus, receptor subunits forming a receptor channel with a linear current-voltage (I-V) relationship (GluR1/2, GluR2/3, and GluR6) were not affected, while receptor subunits with rectifying I-V relationships (GluR1, GluR3, GluR4, and GluR1/3) were reversibly blocked by JSTX. By using receptor-subunit mutants obtained by site-directed mutagenesis, we have identified a single amino acid position (glutamine in the proposed second transmembrane domain) that is critical for the JSTX block. Since this site has previously been shown to control the I-V relationship of the AMPA/KA receptor channel and to participate in the regulation of the channel's permeability for calcium ions, our findings suggest that JSTX binds close to the central pore region of the channel.