Charybdotoxin block of a Shaker K+ channel was studied in Xenopus oocyte macropatches. Toxin on rate increases linearly with toxin concentration in an ionic strength-dependent fashion and is competitively diminished by tetraethylammonium. On rate is insensitive to transmembrane voltage and to K+ on the opposite side of the membrane. Conversely, toxin off rate is insensitive to toxin concentration, ionic strength, and added tetraethylammonium but is enhanced by membrane depolarization or K+ (or Na+) in the trans solution. Charge neutralization of charybdotoxin Lys27, however, renders off rate voltage insensitive. Our results argue that block of voltage-gated K+ channels results from the binding of one toxin molecule, so that Lys27 enters the pore and interacts with K+ (or Na+) in the ion conduction pathway.