Charybdotoxin (CTX) is a peptide of known structure that inhibits Shaker K+ channels by a pore-blocking mechanism. Point mutagenesis of all 30 solvent-exposed residues identified the part of the CTX molecular surface making contact with the receptor in the K+ channel. All close-contact residues are clustered in a well-defined interaction surface; the shape of this surface implies that the outer opening of the Shaker channel conduction pore abruptly widens to a 25 x 35 A plateau. A mutagenic scan of the S5-S6 linker sequence of the Shaker K+ channel identified those channel residues influencing CTX binding affinity. The Shaker residues making the strongest contribution to toxin binding are located close to the pore-lining sequence, and more distant residues on both sides of this region influence CTX binding weakly, probably by an electrostatic mechanism. Complementary mutagenesis of both CTX and Shaker suggests that Shaker-F425 contacts a specific area near T8 and T9 on the CTX molecular surface. This contact point constrains Shaker-F425 to be located at a 20 A radial distance from the pore axis and 10-15 A above the "floor" of the CTX receptor.