We have recently cloned the oc-subunit of a bovine amiloride-sensitive Nachannel (abENaC; Fuller el al. Am. J. Physiol. 269, C641, 1995). This subunit shares extensive homology with both rat and human aENaC 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, the abENaC Nachannel exhibits a main transition to 39 pS, with rare 13 pS step transitions to one of two subconductance states (26 pS and 13 pS). In contrast, arENaC Nachannels have a main transition step to 13 pS, with a second stepwise transition of 26 pS to 39 pS. A deletion mutant of abENaC, encompassing the entire C terminal region (R567X), converts the kinetic behavior of abENaC to that of arENaC, 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 abENaC gating pattern, whereas treatment with the reducing agent dithiothreitol produces only 13 pS transitions. These results are consistent with the hypothesis that disulftde bond formation between the carboxy termini of abENaC subunits account for the different kinetic behavior of this member of the ENaC family of Nachannels. Supported by N1H Grant DK 37206.