The activation of Shaker K+ channels is steeply voltage dependent. To determine whether conserved charged amino acids in putative transmembrane segments S2, S3, and S4 contribute to the gating charge of the channel, the total gating charge movement per channel was measured in channels containing neutralization mutations. Of eight residues tested, four contributed significantly to the gating charge: E293, an acidic residue in S2, and R365, R368, and R371, three basic residues in the S4 segment. The results indicate that these residues are a major component of the voltage sensor. Furthermore, the S4 segment is not solely responsible for gating charge movement in Shaker K+ channels.