To better understand how renal Na⁺ reabsorption is altered by heavy metal poisoning, we examined the effects of several divalent heavy metal ions (Zn²⁺, Ni²⁺, Cu²⁺, Pb²⁺, Cd²⁺ and Hg²⁺) on the activity of single ENaC channels in a renal epithelial cell line (A6). None of the cations changed the single channel conductance. However, ENaC activity (measured as the number of channel times open probability) was decreased by Cd²⁺ and Hg²⁺, increased by Cu²⁺, Zn²⁺ and Ni²⁺, but was unchanged by Pb²⁺. Of the cations that induced an increase in Na channel function, Zn²⁺ increased the number of channels (N) in a patch, Ni²⁺ increased the channel open probability (Po), and Cu²⁺ increased both. The cysteine modification reagent, [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), also increased N; whereas, diethyl pyrocarbonate (DEPC), which covalently modifies histidine residues, has effect neither on channel Po nor on channel N. Cu²⁺ increased channel number and stimulated channel Po by reduction in Na⁺ self inhibition. Furthermore, we observed that the activity of ENaC is slightly voltage dependent, and that voltage dependence of ENaC is insensitive to extracellular Na⁺ concentration; however, apical application of Ni²⁺ or DEPC reduced the channel voltage dependence. Thus, the voltage sensor of Xenopus ENaC is different from that of typical voltage gated channels since voltage appears to be sensed by histidine residues in the extracellular loops (ECLs) of ENaC rather than by charged amino acids located in a transmembrane domain.