Compounds which modulate AMPA receptor function through allosteric mechanisms were examined for their effect on the binding of the agonist [3H]fluorowillardiine (FW). Benzamide-type positive modulators (ampakinestrade mark) under all experimental circumstances increased [3H]FW binding to native receptors in rat brain membranes. Benzothiadiazide drugs had more variable effects ranging from large reductions with cyclothiazide and JM-13 to increases produced by more recent compounds like PEPA, D1 and LY392098. These effects on binding were moderately influenced by the assay conditions, including temperature and the presence or absence of thiocyanate. Significant changes in agonist binding were also produced by other modulatory agents such as noncompetitive blockers (GYKI 53655, SYM 2206), polycationic compounds (spermine, Naspm, philanthotoxin) and polyanionic compounds (Evans Blue, suramin, PPNDS). EC50 values usually were similar to those from physiological studies, which validates using binding tests to assess drug potencies. Moreover, direction and magnitude of the binding change (Emax) provide information about which kinetic aspects are affected by a drug. For example, the magnitude of the binding increase produced by positive modulators was strongly correlated with their ability to slow response deactivation in excised patch recordings. Binding also provides a reliable method to examine whether interactions between agents are competitive. Thus, thiocyanate did not significantly influence the EC50 of cyclothiazide, suggesting distinct sites of action. Taken together, [3H]FW binding can yield important information about drug-receptor and drug-drug interactions for a wide range of modulatory agents. One potential limitation of [3H]FW is a large preference for subunits GluR1 and GluR2 (KD 4-10 nM) over GluR3 and GluR4 (160-600 nM) which implies that tests with brain membranes preferentially reveal drug effects produced at the former two subunits. Lastly, data are shown which highlight the importance of optimizing experimental conditions in filtration assays, for instance by always including thiocyanate in wash buffers.