Slow activation and rapid C-type inactivation produce inward rectification of the current-voltage relationship for human ether-a'-go-go-related gene (hERG) channels. To characterize the voltage sensor movement associated with hERG activation and inactivation, we performed an Ala scan of the 32 amino acids (Gly(514)-Tyr(545)) that comprise the S4 domain and the flanking S3-S4 and S4-S5 linkers. Gating and ionic currents of wild-type and mutant channels were measured using cut-open oocyte Vaseline gap and two microelectrode voltage clamp techniques to determine the voltage dependence of charge movement, activation, and inactivation. Mapping the position of the charge-perturbing mutations (defined as |DeltaDeltaG| > 1.0 kcal/mol) on a three-dimensional S4 homology model revealed a spiral pattern. As expected, mutation of these residues also altered activation. However, mutation of residues in the S3-S4 and S4-S5 linkers and the C-terminal end of S4 perturbed activation (|DeltaDeltaG| > 1.0 kcal/mol) without altering charge movement, suggesting that the native residues in these regions couple S4 movement to the opening of the activation gate or stabilize the open or closed state of the channel. Finally, mutation of a distinct set of residues impacted inactivation and mapped to a single face of the S4 helix that was devoid of activation-perturbing residues. These results define regions on the S4 voltage sensor that contribute differentially to hERG activation and inactivation gating.