Gating differences occur between the alpha-subunits of the bovine and rat clones of an amiloride-sensitive epithelial Na+ channel (ENaC). Deletion of the carboxy terminus of bovine alpha-ENaC (alpha-bENaC) at R567 converted the gating properties to that of rat alpha-ENaC (alpha-rENaC). The equivalent truncation in alpha-rENaC was without effect on the gating of the rat homologue. The addition of actin to ENaC channels composed of either alpha-rENaC or alpha-bENaC alone produced a twofold reduction in conductance and an increase in open probability. Neither alpha-rENaC (R613X) nor alpha-bENaC (R567X) was responsive to actin. Using a chimera consisting of alpha-rENaC1-615 and alpha-bENaC570-650, we examined several different carboxy-terminal truncation mutants plus and minus actin. When incorporated into planar bilayers, the gating pattern of this construct was identical to wild-type (wt) alpha-bENaC. Premature stop mutations proximal to E685X produced channels with gating patterns like alpha-rENaC. Actin had no effect on the E631X truncation, whereas more distal truncations all interacted with actin, as did wt alpha-bENaC. Key findings were confirmed using channels expressed in Xenopus oocytes and studied by cell-attached patch-clamp recording. Our results suggest that the site of actin regulation at the carboxy terminus of the chimera is located between residues 631 and 644.