Site-directed mutagenesis and the two-electrode voltage-clamp techniques were used to evaluate the site of action of picrotoxin on rat alpha 1 beta 2 gamma 2 containing GABAA receptors expressed in Xenopus oocytes. Following a sequence comparison between GABAA subunits and the picrotoxin-insensitive glycine beta subunit, the following mutations were made near the center of the M2 region of the alpha 1, beta 2, and gamma 2 GABAA subunits: alpha 1(T261F/T267A), beta 2(T246F/T252A), and gamma 2(T271F/T277A). Wild type (alpha 1 beta 2 gamma 2) GABA channels had an IC50 for picrotoxin of 1.3 +/- O.3 microM. In contrast, alpha 1 beta 2 gamma 2 channels that contained any one of the mutated alpha 1, beta 2, or gamma 2 subunits produced currents that were insensitive to picrotoxin (0.1-100 microM). The single mutant beta 2(T246F), in combination with wild type alpha and gamma subunits, also conferred picrotoxin-insensitivity. In contrast, combinations containing beta 2(T252A) were blocked by picrotoxin with an IC50 of 1.4 +/- 0.4 microM. In some instances, the EC50 to GABA was slightly altered in the mutant receptors; but no change was observed in EC50 or potentiation by the allosteric modulator, alprazolam. The data in this study suggest that picrotoxin's site of action is within the channel pore; however the mechanism by which picrotoxin blocks current remains unknown.