GIRK channels regulate membrane excitability dependent on GPCR activity. Gain-of-function mutations in the channels have been identified in patients suffering from several diseases; e.g. primary aldsteronism. Since gene elimination of these channels yielded mild phenotypes of the mice, the channel blockage could be expected as therapeutic strategy. While channel blockers used to bind at a central cavity of ion permeation pathway located at the membrane domain, a loose spatial constraint of the mode of binding leads to a low selectivity of drug-channel interaction. Wide distribution of binding sites of their activators and blockers suggests that the prerequisite of conformational changes of entire molecules for functioning and the possibility to design allosteric modulators that bind to sites out of the central cavity. In an electron density map of a high-resolution crystal structure of Kir3.2 cytoplasmic region, we identified an electron density which was not belonging to the polypeptide chain. A model compound related to the shape of the density inhibited Kir3.2 activity. These results strongly suggested that the electron density and its surrounding area correspond to an allosteric modulator and its binding site.