Patch-clamp studies of cytoplasmic drops from the charophyte Chara australis have previously revealed K+ channels combining high conductance (170 pS) with high selectivity for K+, which are voltage activated. The cation-selectivity sequence of the channel is shown here to be: K+ greater than Rb+ greater than NH4+ much greater than Na+ and Cl-. Divalent cytosolic ions reduce the K+ conductance of this channel and alter its K+ gating in a voltage-dependent manner. The order of blocking potency is Ba2+ greater than Sr2+ greater than Ca2+ greater than Mg2+. The channel is activated by micromolar cytosolic Ca2+, an activation that is found to be only weakly voltage dependent. However, the concentration dependence of calcium activation is quite pronounced, having a Hill coefficient of three, equivalent to three bound Ca2+ needed to open the channel. The possible role of the Ca(2+)-activated K+ channel in the tonoplast of Chara is discussed.