Aims: In some patients with atypical cystic fibrosis (CF) only one allele of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is affected. Mutations of the epithelial sodium channel (ENaC) may contribute to the pathophysiology in these patients. The aim of the present study was to functionally characterize a mutation in the b-subunit of ENaC (bV348M) recently identified in a patient with atypical CF (Mutesa et al. 2009, Chest 135, 1233-42). Methods: Wild-type (wt) abgENaC or mutant abV348MgENaC were expressed in Xenopus laevis oocytes and ENaC function was assessed by measuring the amiloride (2 mM) sensitive whole-cell current (DI_ami) with two-electrode voltage-clamp. The well characterized bS520C mutation was used as a tool to obtain channels that can be converted to channels with an open probability (Po) of close to one upon application of the sulfhydryl reagent MTSET (1 mM). n indicates individual number of oocytes, N indicates number of batches of oocytes. Results: Baseline DI_ami was ~40% higher in mutant (n=191) than in wt ENaC expressing oocytes (n=190, N=19, p<0.001). The stimulatory effect of MTSET on DI_ami was larger in abS520CgENaC expressing oocytes (4.2-fold, n=23) than in abV348M-S520CgENaC expressing oocytes (3-fold, n=23, N=3, p<0.001). Similarly, proteolytic activation by chymotrypsin (2 mg/ml) was larger in wt ENaC (3.8-fold, n=24) than in mutant ENaC (2.9-fold, n=24, N=3, p<0.001). The stimulatory effect of the ENaC activator S3969 (10 mM) was also larger on wt ENaC (3.4-fold, n=24) than on mutant ENaC (2.5-fold, n=24, N=3; p<0.001). Conclusion: The increased baseline activity of the mutant channel and its reduced responsiveness to stimulation indicate that the mutation increases average channel Po. This gain-of-function effect of the bV348M mutation may contribute to CF pathophysiology by inappropriately increasing sodium and fluid absorption in the respiratory tract.