Guanyl nucleotides inhibit the binding of the AMPA receptor agonists [3H]fluorowillardiine and [3H]AMPA and the competitive antagonist [3H]CNQX to rat brain cerebrocortical membranes. The rank order of inhibition for each of the radioligands tested was GTP > GDP > GMP. The nucleotides CTP and ATP showed no effect. GTP inhibition was unaffected by the presence or absence of NaCl and MgCl2. Pre-treatment of the membranes with GTP, and its removal before addition of radioligand, did not inhibit binding. Quantitative autoradiography demonstrated that GTP inhibition occurred throughout the brain. These results are consistent with guanyl nucleotides acting at an extracellular site present on all AMPA receptor subunits, occupation of which inhibits agonist and antagonist binding.