The gating of Ca2+-activated Cl− channels is controlled by a complex interplay among [Ca2+]i, membrane potential and permeant anions. Besides Ca2+, Ba2+ also can activate both TMEM16A and TMEM16B. This study reports the effects of several divalent cations as regulators of TMEM16A channels stably expressed in HEK293T cells. Among the divalent cations that activate TMEM16A, Ca2+ is most effective, followed by Sr2+ and Ni2+, which have similar affinity, while Mg2+ is ineffective. Zn2+ does not activate TMEM16A but inhibits the Ca2+-activated chloride currents. Maximally effective concentrations of Sr2+ and Ni2+ occluded activation of the TMEM16A current by Ca2+, which suggests that Ca2+, Sr2+ and Ni2+ all regulate the channel by the same mechanism.