An increased activity of the epithelial sodium channel (ENaC) in the airways may contribute to the pathophysiology of cystic fibrosis (CF), an autosomal recessive disorder caused by mutations in the CFTR gene. In some atypical CF patients a mutation can be identified in only one CFTR allele. In this group of patients we recently identified a heterozygous mutation (F61L) in the a-subunit of ENaC. To investigate the functional effect of this mutation, we expressed human wild-type aßg-ENaC or mutant a_F61Lßg-ENaC in Xenopus laevis oocytes. Two-electrode voltage-clamp experiments demonstrated a 90% reduction of amiloride-sensitive whole cell currents (DI_ami) in mutant ENaC expressing oocytes. Western blot analysis confirmed that whole cell protein expression levels of wild-type and mutant a-ENaC were similar. However, surface expression of the mutant channel, detected with a chemiluminescence assay, was reduced by about 40%. Experiments using the ßS520C mutant ENaC, which can be converted to a channel with an open probability of nearly one by exposure to a sulfhydryl reagent, demonstrated a 70% reduction of the average open probability (P_o) of the mutant channel. This was confirmed by our finding that the stimulatory effect of chymotrypsin on DI_ami was higher in mutant ENaC expressing oocytes than in wild-type expressing oocytes. Moreover, Na⁺ self inhibition, a mechanism which inhibits ENaC within seconds when the extracellular Na⁺ concentration is acutely increased, was enhanced in the mutant channel. In summary, the aF61L mutation leads to a decrease of channel activity. Loss-of-function mutations of ENaC have previously been shown to cause pseudo-hypoaldosteronism type I (PHA1), a disease characterized by renal salt wasting. In addition, patients with PHA1 have been described to suffer from respiratory symptoms similar to those of patients with atypical CF. In PHA1 a reduction of ENaC-mediated Na⁺ absorption in respiratory epithelia is thought to favour fluid secretion in the respiratory tract. Our finding that the F61L mutation impairs ENaC function suggests that the mutation may contribute to respiratory symptoms in atypical CF patients with this mutation.