Large-conductance Ca²⁺-activated K⁺ channels can be activated by membrane voltage in the absence of Ca²⁺ binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca²⁺-activated K⁺ channels in the virtual absence of Ca²⁺ (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge–voltage relationship (Q–V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G–V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (τ_ON = 60 μs at +200 mV, τ_OFF = 16 μs at −80 mV). However, Q_OFF increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I_K activation. The slow onset of this gating charge prevents its detection as a component of I_gON, although it represents ∼40% of the total charge moved at +140 mV. The decay of I_gOFF is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277–304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C–C, O–O, and C–O transitions.