Abstract We have determined the binding site on agitoxin2 (AgTx2) to the KcsA K + channel by a transferred cross-saturation (TCS) experiment. The residues significantly affected in the TCS experiments formed a contiguous surface on AgTx2, and substitutions of the surface residues decreased the binding affinity to the KcsA K + channel. Based on properties of the AgTx2 binding site with the KcsA K + channel, we present a surface motif that is observed in pore-blocking toxins affecting the K + channel. Furthermore, we also explain the structural basis of the specificity of the K + channel to the toxins. The TCS method utilized here is applicable not only for the channels, which are complexed with other inhibitors, but also with a variety of regulatory molecules, and provides important information about their interface in solution.