The epithelial Na⁺ channel (ENaC) is modulated by various extracellular factors including Na⁺, organic or inorganic cations and serine proteases. To identify the effect of the divalent nickel cation on ENaC we compared Na⁺ permeability and amiloride kinetics of xENaC and rENaC heterologously expressed in Xenopus laevis oocytes. We found that the channel cloned from the kidney of the clawed toad Xenopus laevis (wild-type (WT) xENaC) was stimulated by external Ni²⁺, whereas the divalent cation inhibited the channel cloned from the rat colon (wild-type rENaC). Kinetics of amiloride binding was determined by noise analysis of blocker-induced fluctuation in current adapted for the transoocyte voltage-clamp (TOVC) method, and Na⁺ conductance was assessed with the dual electrode voltage clamp (TEVC). The inhibitory effect of Ni²⁺ on amiloride binding is not species dependent as Ni²⁺ decreased the affinity (mainly reducing the association rate constant) of the blocker in both species, in competition with Na⁺. Importantly, we found a prominent difference in channel conductance at hyperpolarizing voltage pulses using TEVC. In wild-type xENaC, the initial ohmic current response was stimulated by Ni²⁺ whereas the secondary voltage-activated current component remained unaffected. In wild-type rENaC only a voltage-dependent block by Ni²⁺ was obtained. To further study the origin of the xENaC stimulation by Ni²⁺, and based on the rationale of the well-known high affinity of Ni²⁺ for histidine residues, we designed subunit mutants of xENaC, by substituting histidines, that were expressed in oocytes together with wild-type and subunits. Neither changing H²¹⁵ to Asp in one putative amiloride-binding domain (WYRFHY) in the extracellular loop, between Na⁺ channel membrane segments M1 and M2, had an influence on the stimulatory effect of Ni²⁺, nor did complete deletion of this segment. Next, we mutated H⁴¹⁶, flanked by H⁴¹¹ and C⁴¹⁷, a unique site for possible heavy metal ion chelation and -with this quality- most proximal (about 100 amino acids upstream of the second putative amiloride binding site at the pore entrance) localized at M2. Replacing H⁴¹⁶ by arginine, aspartic acid, tyrosine and alanine clearly affected amiloride binding in all cases and also Na⁺ conductance, as expressed in the xENaC current-voltage relation, especially with aspartate and tyrosine. However, none of these mutations, and so like those obtained with the WYRFHY stretch, could abolish the stimulating effect of Ni²⁺ or reverse it to an inhibitory type.