We have recently cloned the α subunit of a bovine amiloride-sensitive Na+ channel (αbENaC). This subunit shares extensive homology with both rat and human αENaC subunits but shows marked divergence at the C terminus beginning at amino acid 584 of the 697-residue sequence. When incorporated into planar lipid bilayers, αbENaC almost exclusively exhibits a main transition to 39 picosiemens (pS) with very rare 13 pS step transitions to one of two subconductance states (26 and 13 pS). In contrast, the α subunit of the rat renal homolog of ENaC (αrENaC) has a main transition step to 13 pS that is almost constituitively open, with a second stepwise transition of 26 to 39 pS. A deletion mutant of αbENaC, encompassing the entire C- terminal region (R567X), converts the kinetic behavior of αbENaC to that of αrENaC, i.e. a transition to 13 pS followed by a second 26 pS transition to 39 pS. Chemical cross-linking of R567X restores the wild-type αbENaC gating pattern, whereas treatment with the reducing agent dithiothreitol produced only 13 pS transitions. In contrast, an equivalent C-terminal truncation of αerENaC (R613X) had no effect on the gating pattern of αrENaC. These results are consistent with the hypothesis that interactions between the C termini of αbENaC account for the different kinetic behavior of this member of the ENaC family of Na+ channels.