The delta-subunit of epithelial Na(+) channels (ENaC) is predominately expressed in brain, heart, and pancreas. The amiloride sensitivity, Na(+) conductance, and critical domains for gating are characterized as a cross between proton-activated Na(+) channels and alpha-ENaC. The hypothesis that external protons may activate human delta-ENaC was addressed by expressing deltabetagamma-hENaC in Xenopus oocytes and evaluating proton-activated current with the two-electrode voltage clamp technique. Our results showed that protons transiently evoked a Na(+) current with an EC(50) of pH 6 overlapped on the basal current of deltabetagamma-hENaC. Proton-activated current was not observed in uninjected oocytes. Studies on gating kinetics revealed that activation, desensitization, and recovery times of proton-activated Na(+) current were 3.8 +/- 0.5 s, 253 +/- 9.5 s, and 10 +/- 3.6 s, respectively (n = 4-12). Alkali metal cation selectivity of the proton-activated current was identical to that of the basal current of deltabetagamma-hENaC. The metabolic acids, lactate, pyruvate, and formate, modified the proton-activated current, as did hypo-osmotic stress. EDTA, hypo-osmolarity, and lactate enhanced proton activation synergistically. Our results suggest that delta-hENaC subunit is essential for proton-activated current and gamma-subunit may potentially regulate the response of delta-hENaC to protons. We have concluded that deltabetagamma-hENaC is a proton-activated cation channel whose closing gate can be regulated by a proton-induced conformational change. Proton-sensitivity of deltabetagamma-hENaC may be an important mechanism for integrating external ischemic signals in inflamed and hypoxic tissues.