Voltage-activated ion channels respond to changes in membrane voltage by coupling the movement of charges to channel opening. A K⁺ channel-specific radioligand was designed and used to determine the origin of these gating charges in the Shaker K⁺ channel. Opening of a Shaker K⁺ channel is associated with a displacement of 13.6 electron charge units. Gating charge contributions were determined for six of the seven positive charges in the S4 segment, an unusual amino acid sequence in voltage-activated cation channels consisting of repeating basic residues at every third position. Charge-neutralizing mutations of the first four positive charges led to large decreases (∼4 electron charge units each) in the gating charge; however, the gating charge of Shaker Δ10, a Shaker K⁺ channel with 10 altered nonbasic residues in its S4 segment, was found to be identical to the wild-type channel. These findings show that movement of the NH₂-terminal half but not the CO₂H-terminal end of the S4 segment underlies gating charge, and that this portion of the S4 segment appears to move across the entire transmembrane voltage difference in association with channel activation.