Epithelial Na(+) channel (ENaC) activity is regulated, in part, by the cortical cytoskeleton. Here we demonstrate that cortactin is highly expressed in the kidney cortex and polarized epithelial cells, and is localized to the cortical collecting duct. Coexpression of cortactin with ENaC decreases ENaC activity, as measured in patch-clamp experiments. Biotinylation experiments and single-channel analysis reveal that cortactin decreases ENaC activity via affecting channel open probability (P(o)). Knockdown of cortactin in mpkCCD(c14) principal cells results in an increase in ENaC activity and sodium reabsorption. Coimmunoprecipitation analysis shows direct interactions between cortactin and all three ENaC subunits in cultured and native cells. To address the question of what mechanism underlies the action of cortactin on ENaC activity, we assayed the effects of various mutants of cortactin. The data show that only a cortactin mutant unable to bind Arp2/3 complex does not influence ENaC activity. Furthermore, inhibitor of the Arp2/3 complex CK-0944666 precludes the effect of cortactin. Depolymerization of the actin microfilaments and inhibition of the Arp2/3 complex does not result in the loss of association between ENaC and cortactin. Thus, these results indicate that cortactin is functionally important for ENaC activity and that Arp2/3 complex is involved in this mechanism.