To determine how intracellular Ca²⁺ and membrane voltage regulate the gating of large conductance Ca²⁺-activated K⁺ (BK) channels, we examined the steady-state and kinetic properties of mSlo1 ionic and gating currents in the presence and absence of Ca²⁺ over a wide range of voltage. The activation of unliganded mSlo1 channels can be accounted for by allosteric coupling between voltage sensor activation and the closed (C) to open (O) conformational change (Horrigan, F.T., and R.W. Aldrich. 1999. J. Gen. Physiol. 114:305–336; Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277–304). In 0 Ca²⁺, the steady-state gating charge-voltage (Q_SS-V) relationship is shallower and shifted to more negative voltages than the conductance-voltage (G_K-V) relationship. Calcium alters the relationship between Q-V and G-V, shifting both to more negative voltages such that they almost superimpose in 70 μM Ca²⁺. This change reflects a differential effect of Ca²⁺ on voltage sensor activation and channel opening. Ca²⁺ has only a small effect on the fast component of ON gating current, indicating that Ca²⁺ binding has little effect on voltage sensor activation when channels are closed. In contrast, open probability measured at very negative voltages (less than −80 mV) increases more than 1,000-fold in 70 μM Ca²⁺, demonstrating that Ca²⁺ increases the C-O equilibrium constant under conditions where voltage sensors are not activated. Thus, Ca²⁺ binding and voltage sensor activation act almost independently, to enhance channel opening. This dual-allosteric mechanism can reproduce the steady-state behavior of mSlo1 over a wide range of conditions, with the assumption that activation of individual Ca²⁺ sensors or voltage sensors additively affect the energy of the C-O transition and that a weak interaction between Ca²⁺ sensors and voltage sensors occurs independent of channel opening. By contrast, macroscopic I_K kinetics indicate that Ca²⁺ and voltage dependencies of C-O transition rates are complex, leading us to propose that the C-O conformational change may be described by a complex energy landscape.