Cystic fibrosis (CF) airway epithelia exhibit enhanced Na+ reabsorption in parallel with diminished Cl- secretion. We tested the hypothesis that actin plays a role in the regulation of a cloned epithelial Na+ channel (ENaC) by the cystic fibrosis transmembrane conductance regulator (CFTR). We found that immunopurified bovine tracheal CFTR coreconstituted into a planar lipid bilayer with α,β,γ-rat ENaC (rENaC) decreased single-channel open probability (P(o)) of rENaC in the presence of actin by over 60%, a significantly greater effect than was observed in the absence of actin ( 1/4 0%). In the presence of actin, protein kinase A plus ATP activated both CFTR and rENaC, but CFTR was activated in a sustained manner, whereas the activation of rENaC was transitory. ATP alone could also activate ENaC transiently in the presence of actin but had no effect on CFTR. Stabilizing short actin filaments at a fixed length with gelsolin (at a ratio to actin of 2:1) produced a sustained activation of α,β,γ-rENaC in both the presence or absence of CFTR. Gelsolin alone (i.e., in the absence of actin) had no effect on the conductance or P(o) of either CFTR or rENaC. We have also found that short actin filaments produced their modulatory action on α-rENaC independent of the presence of the β- or γ-rENaC subunits. In contrast, CFTR did not affect any properties of the channel formed by α-rENaC alone, i.e., in the absence of β- or γ-rENaC. These results indicate that CFTR can directly downregulate single Na+ channel activity, which may account for the observed differences between Na+ transport in normal and CF-affected airway epithelia. Moreover, the presence of actin confers an enhanced modulatory ability of CFTR on Na+ channels.