The activation of a calcium-dependent K+ channel K(Ca) in cultured hippocampal neurons has been studied after the addition, to the internal solution, of the divalent cations magnesium (Mg2+), strontium (Sr2+) or barium (Ba2+). With physiological K+ across inside-out patches and Ca2+ present at 0.2 mM in the bath solution, a 90-pS channel was activated with an open probability in excess of 75%. When the internal Ca2+ was reduced to levels near 5 microM, the channel-open probability was significantly diminished. However, if 0.2 mM concentrations of either Mg2+ or Sr2+ were added to the internal solution, the open probability was increased to a value close to original level. In the presence of internal Ca2+ at 0.1 microM, the K(Ca) channel was not active and was not activated with the addition of 0.2 mM Mg2+ or Sr2+ to the internal solution. Thus, Mg2+ or Sr2+ were not able to active K(Ca) in the absence of Ca2+; however, both of these divalent cations could potentiate the Ca(2+)-induced activation of K(Ca) if internal Ca2+ was near 5 microM. The results indicate that Mg2+ could have a role as an internal modulator of K(Ca) in the Ca(2+)-dependent regulation of excitability in nerve membrane. Replacement of Ca2+ with Ba2+, or addition of Ba2+ to Ca-containing solutions, caused significant decreases in the channel-open probability for K(Ca). The action of Ba2+ was primarily mediated by a decrease in the frequency of channel opening. At a concentration of 5 microM, Ba2+ diminished the channel-open probability by one-half.