Hanatoxin (HaTx) binds to multiple sites on the surface of the drk1 voltage-gated K+ channel and modifies channel gating. We set out to identify channel residues that contribute to form these HaTx binding sites. Chimeras constructed using the drk1 and shaker K+ channels suggest that the S3-S4 linker may contain influential residues. Alanine scanning mutagenesis of the region extending from the C terminal end of S3 through S4 identified a number of residues that likely contribute to form the HaTx binding sites. The pore blocker Agitoxin2 and the gating modifier HaTx can simultaneously bind to individual K+ channels. These results suggest that residues near the outer edges of S3 and S4 form the HaTx binding sites and are eccentrically located at least 15 A from the central pore axis on the surface of voltage-gated K+ channels.