The epithelial sodium channel (ENaC) and the CFTR chloride channel critically regulate airway surface liquid by driving fluid absorption and secretion, respectively. Their functional interplay is complex and incompletely understood. ENaC is a heteromeric channel with three well characterised subunits (α, β, ). In humans, an additional ENaC subunit exists in lung and several other tissues where it may replace the α-subunit to form βENaC. Little is known about the physiological role of βENaC and its possible interaction with CFTR. The aim of the present study was to investigate the effect of human CFTR on human βENaC heterologously expressed in Xenopus laevis oocytes. In oocytes co-expressing βENaC and CFTR the ENaC mediated amiloride-sensitive whole-cell current (I_ami) was reduced by ~50% compared to that measured in oocytes expressing βENaC alone. Moreover, basal level of proteolytic ENaC activation was reduced in the presence of CFTR. The inhibitory effect of CFTR on βENaC was due to a combination of decreased average open probability (Po) and reduced channel expression at the cell surface. Interestingly, in oocytes expressing βENaC increasing intracellular [cAMP] by IBMX and forskolin increased I_ami by ~50%. This stimulatory effect was not observed for human and rat αβENaC and was independent of CFTR co-expression and co-activation. Experiments with a mutant channel (β_S520CENaC) which can be converted to a channel with a Po of nearly one suggested that cAMP activates βENaC by increasing Po. In conclusion our results demonstrate that βENaC is inhibited by CFTR but activated by cAMP.