The S4 transmembrane segment is the primary voltage sensor in voltage-dependent ion channels. Its movement in response to changes in membrane potential leads to the opening of the activation gate, which is formed by a separate structural component, the S6 segment. Here we show in voltage-, Ca 2+ -, and Mg 2+ -dependent, large conductance K + channels that the S4 segment participates not only in voltage- but also Mg 2+ -dependent activation. Mutations in S4 and the S4-S5 linker alter voltage-dependent activation and have little or no effect on activation by micromolar Ca 2+ . However, a subset of these mutations in the C-terminal half of S4 and in the S4-S5 linker either reduce or abolish the Mg 2+ sensitivity of channel gating. Cysteine residues substituted into positions R210 and R213, marking the boundary between S4 mutations that alter Mg 2+ sensitivity and those that do not, are accessible to a modifying reagent [sodium (2-sulfonatoethyl)methane-thiosulfonate] (MTSES) from the extracellular and intracellular side of the membrane, respectively, at -80 mV. This implies that interactions between S4 and a cytoplasmic domain may be involved in Mg 2+ -dependent activation. These results indicate that the voltage sensor is critical for Mg 2+ -dependent activation and the coupling between the voltage sensor and channel gate is a converging point for voltage- and Mg 2+ -dependent activation pathways.