In some patients with atypical cystic fibrosis (CF), only one allele of the CF transmembrane conductance regulator (CFTR) gene is affected. Mutations of the epithelial sodium channel (ENaC) may contribute to the pathophysiology of the disease in these patients. To functionally characterize a mutation in the β-subunit of ENaC (βV348M) recently identified in a patient with severe CF-like symptoms (Mutesa et al. 2009), we expressed wild-type (wt) αβγENaC or mutant αβV348MγENaC in Xenopus laevis oocytes. The βV348M mutation stimulated amiloride-sensitive whole-cell current (ΔI(ami)) by ∼40% but had no effect on surface expression or single-channel conductance of ENaC. Instead the mutation increased channel open probability (P(o)). Proteolytic activation of mutant ENaC by chymotrypsin was reduced compared with that of wt ENaC (∼3.0-fold vs. ∼4.2-fold), which is consistent with the increased baseline P(o) of mutant ENaC. Similarly, the ENaC activator S3969 stimulated mutant ENaC currents to a lesser degree (by ∼2.6-fold) than wt ENaC currents (by ∼3.5-fold). The gain-of-function effect of the βV348M mutation was confirmed by whole-cell current measurements in HEK293 cells transiently transfected with wt or mutant ENaC. Computational channel modeling in combination with functional expression of different βV348 mutants in oocytes suggests that the βV348M mutation increases channel P(o) by destabilizing the closed channel state. Our findings indicate that the gain-of-function effect of the βV348M mutation may contribute to CF pathophysiology by inappropriately increasing sodium and fluid absorption in the respiratory tract.