Agonist responses for flip splice variants of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR-A, -C, and -D are more strongly potentiated by cyclothiazide than are those for the flop forms. Cyclothiazide shows both greater efficacy and higher apparent affinity for potentiation of GluR-Aflip versus GluR-Aflop. Consistent with higher affinity for the flip splice variant, recovery from potentiation by cyclothiazide proceeds 30 times more slowly for GluR-Aflip than for GluR-Aflop. In the presence of 300 microM cyclothiazide a 6-fold leftward shift in the kainate dose-response curve for GluR-Aflip but not GluR-Aflop additionally contributes to a difference in potentiation for these splice variants. Although control responses to glutamate show strong desensitization for both splice variants of GluR-A, in the presence of 100 microM cyclothiazide desensitization is strongly attenuated for GluR-Aflip, whereas for GluR-Aflop desensitization remains pronounced but with a rate of onset slowed 50-fold, compared with control. In heteromeric AMPA receptors formed from GluR-A and GluR-B, the flip splice variants are dominant for controlling both recovery from potentiation of responses to kainate and block of desensitization of responses to glutamate. Our results suggest that the flip/flop module could directly contribute to the binding site for cyclothiazide, raising the possibility that this site is located in an extracellular receptor domain.