The activation of BK type Ca2+-activated K+ channels depends on both voltage and Ca2+. We studied three point mutations in the putative voltage sensor S4 or S4-S5 linker regions in the mslol BK channels to explore the relationship between voltage and Ca2+ in activating the channel. These mutations reduced the steepness of the open probability - voltage (P(o) - V) relation and increased the shift of the P(o) - V relations on the voltage axis in response to increases in the calcium concentration. It is striking that these two effects were reciprocally related for all three mutations, despite different effects of the mutations on other aspects of the voltage dependence of channel gating. This reciprocal relationship suggests strongly that the free energy contributions to channel activation provided by voltage and by calcium binding are simply additive. We conclude that the Ca2+ binding sites and the voltage sensors do not directly interact. Rather they both affect the mslol channel opening through an allosteric mechanism, by influencing the conformational change between the closed and open conformations. The mutations changed the channel's voltage dependence with little effect on its Ca2+ affinitiy.