The effects of different intracellular divalent cations on the gating of single batrachotoxin-activated Na+ channels were investigated in planar lipid bilayers. Intracellular divalent cations increased the open probability (Po) of Na+ channels; the gating curve [Po versus membrane potential (Vm) relationship] shifted to more negative potentials. The relative ability of different intracellular divalent cations in shifting the gating curve decreased in the sequence: Mg2+, Ca2+, Ba2+, Sr2+. The cations Ca2+, Ba2+, and Sr2+ induced a larger voltage shift when applied to the extracellular than to the intracellular side of the Na+ channel, whereas, Mg2+ induced the same voltage shift from both sides. The increase in Po induced by intracellular divalent cations was the result of a simultaneous decrease in the closing rate and increase in the opening rate constant, however, the effect of intracellular divalent cations on the closing rate was larger than on the opening rate. These results suggest that there are both differences in surface charge densities between the intracellular and extracellular surfaces of the Na+ channel and differences in chemical affinities of those charges for different divalent cations. The effects of internal divalent cations on Na+ channel gating cannot be explained solely by surface charge reduction, which predicts that the opening and closing rates should be affected equally, but rather are consistent with a mechanism that involves screening and binding of surface charges present on the channel, plus a specific modulatory effect that accounts for the preferential effect of intracellular divalent cations on the closing rate constant.