Native AMPA receptor complexes in the CNS are composed of hetero-oligomers of the GluR1-4 subunits, and generally contain the GluR2 subunit. To determine the contributions of GluR2 to pharmacological properties of receptor complexes, the effect of hetero-oligomerization with GluR2 on allosteric modulation of recombinant AMPA receptors was studied. The study of homo-oligomeric GluR2 was facilitated with a site-directed mutant of the pore, GluR2(R607Q), which allowed robust currents from this normally low-conducting subunit. The efficacy of the allosteric modulators was tested on homo-oligomeric GluR1-4, and then compared with hetero-oligomeric GluR1/GluR2, GluR3/GluR2 and GluR4/GluR2. Two selective allosteric modulators were tested, a positive modulator, cyclothiazide, and a negative modulator, LY300164. The results show that the pharmacological properties of homo-oligomeric GluR2 are not significantly different from those of GluR1, GluR3 or GluR4. The apparent affinity of cyclothiazide is not significantly changed upon hetero-oligomerization. However, the extent of potentiation of kainate responses by cyclothiazide is significantly decreased upon hetero-oligomerization. Hetero-oligomerization increases the apparent affinity of LY300164, a (-) isomer of the 2,3-benzodiazepine LY293606. These data indicate that although GluR2 has a dominant effect on the permeation properties, this subunit does not have a similarly dominant effect on pharmacological properties of native receptors. However, the state of hetero-oligomerization can alter the pharmacological properties of AMPA receptors.