The epithelial sodium channel (ENaC) is a sodium-selective ion channel within the apical membrane of various vertebrate epithelial cells. ENaC consists of three subunits (α, β, γ), however, an additional δ-subunit can replace α-ENaC. We recently demonstrated that incorporation of the δ-subunit into Xenopus ENaC changes channel activity from a constitutively-open into a transient form (1), yet, regulators of this transient activity remain unknown. Based on the evolutionary relationship between δ-ENaC and acid-sensing ion channels within the degenerin/ENaC protein family, we hypothesized that protons regulate the activity of this ENaC isoform. Activity of Xenopus ENaCs was measured by two-electrode voltage-clamp and cell-attached Patch-Clamp electrophysiology using the Xenopus oocyte expression system. Data are presented as mean ± SEM. Reduction of extracellular pH from pH 8.0 to 6.0 increased amiloride-sensitive transmembrane currents of oocytes expressing δβγ-ENaC by a factor of 7.8 ± 0.9 (n=15) whereas currents mediated by αβγ-ENaC were increased by a factor of 1.1 ± 0.04 (n=12; Student’s unpaired t-test: p<0.001). Patch-Clamp recordings indicated that changes in the extracellular pH affected the open probability (Po) of δβγ-ENaC. Compared to channel Po at pH 7.4 (0.28 ± 0.04, n=18) alkaline pH 8.0 reduced Po (0.11 ± 0.02, n=15) whereas acidic pH 6.0 increased Po (0.52 ± 0.04, n=15; ANOVA: p<0.001). Transmembrane currents mediated by δβγ-ENaC are sensitive to sodium self-inhibition (SSI), the fast inhibition of ENaC in the presence of high extracellular [Na+]. Changes in extracellular pH influenced the magnitude of SSI as %-inhibition was maximal at pH 8 (55.7 ± 3.5 %, n=10) and pH 7.4 (53.8 ± 3.5 %, n=17) but declined with extracellular acidification (pH 7: 39.5 ± 4.9 %, n=13; pH 6: 24.7 ± 2.3 % n=10; ANOVA: p<0.001). A pH-sensitive aspartate of a putative Na+-sensing site within the extracellular domain of δβγ-ENaC was replaced by asparagine via site-directed mutagenesis (δD296N). Compared to wildtype δβγ-ENaC (δwt), this mutation had a reduced SSI (%-inhibition of δwt: 54.5 ± 3.4 %, n=18, δD296N: 24.7 ± 1.3 %, n=11; Student’s unpaired t-test: p<0.001) and a reduced maximal pH-mediated channel activation between pH 8-6 (fold-activation of δwt: 5.1 ± 0.7, n=8; δD296N: 2.6 ± 0.2, n=8; Mann-Whitney U-test: p<0.001). Furthermore, human δβγ-ENaC which does not display SSI, showed a markedly reduced fold-activation by extracellular acidification (1.1 ± 0.02, n=12). These results demonstrate that Xenopus δβγ-ENaC is highly sensitive to changes in the extracellular pH whereas channels containing the α-subunit are not. This further supports the hypothesis that Xenopus δ-ENaC represents a functional example for ENaC at the interface of stimulus-activated and constitutively open ion channels in the degenerin/ENaC protein family.