Cystic fibrosis (CF) is an autosomal recessive hereditary disease usually caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A functional interaction of the epithelial sodium channel (ENaC) and the CFTR chloride channel is suspected to play a role in the pulmonary pathophysiology of cystic fibrosis (1). Transgenic mice overexpressing the β-subunit of ENaC in the airways develop a CF-like pulmonary phenotype (2). Interestingly, CF-like pulmonary symptoms have also been reported in patients with pseudohypoaldosteronism type1 (PHA1) caused by loss-of-function mutations in ENaC (3). Thus, increased or decreased ENaC activity in respiratory epithelia may be associated with pulmonary symptoms (4). Recently, several ENaC mutations were identified in patients with atypical CF in whom a mutation could not be identified on both CFTR alleles (5). Interestingly, these mutations include a gain-of-function (W493R) and a loss-of-function mutation (F61L) in the α-subunit of ENaC. To investigate the functional effect of these mutations, we co-expressed the mutant α-subunits together with wild-type β- and γ-subunits in Xenopus laevis oocytes and compared the two mutant channels (α_W493Rβγ or α_F61Lβγ) with wild-type αβγ-ENaC. The W493R mutation stimulated ENaC-mediated whole-cell currents by about fourfold without a concomitant increase in single-channel conductance or channel surface expression. The αF61L mutation reduced ENaC mediated whole-cell currents by about 90%, decreased channel surface expression by about 40%, and did not alter the single-channel conductance. These findings indicate that the mutations mainly affect the average open probability (P_O) of ENaC. This was confirmed by experiments using the βS520C mutant ENaC which can be converted to a channel with a P_O of nearly one, and by experiments using chymotrypsin to proteolytically activate the channel. Interestingly, the ENaC-activator S3969 partially rescued the loss-of-function phenotype of the F61L mutation. We conclude that gain-of-function and loss-of-function mutations may contribute to respiratory symptoms in atypical CF patients and that a mutant channel with a partial loss of function may be amenable to treatment with an ENaC activator.