The hypothesis that 30-amino acid peptides corresponding to the C- terminal portion of the β- and/or γ-rat epithelial sodium channel (rENaC) subunits block constitutively activated ENaC was tested by examining the effects of these peptides on wild-type (wt) rENaC (αβγ-rENaC), truncated Liddle's mutants (αβ(T)γ-, αβγ(T)-, and αβ(T)γ(T)-rENaC), and point mutants (αβ(Y)γ-, αβγ(Y)-rENaC) expressed in Xenopus oocytes. The chord conductances of αβ(T)γ-, αβγ(T)-, and αβ(T)γ(T)-rENaC were 2- or 3- fold greater than for wt αβγ-rENaC. Introduction of peptides into oocytes expressing αβ(T)γ-, αβγ(T)-, and αβ(T)γ(T)-rENaC produced a concentration-dependent inhibition of the amiloride-sensitive Na+ conductances, with apparent dissociation constants (K(d)) ranging from 1700 to 160 μM, depending upon whether individual peptides or their combination was used. Injection of peptides alone or in combination into oocytes expressing wt αβγ-rENaC or single-point mutants did not affect the amiloride-sensitive whole-cell currents. The single channel conductances of all the mutant ENaCs were the same as that of wild type (αβγ-). The single channel activities (N.P(o)) of the mutants were ~2.2-2.6-fold greater than wt αβγ-rENaC (1.08 ± 0.24, n = 7) and were reduced to 1.09 ± 0.17 by 100 μM peptide mixture (n = 9). The peptides were without effect on the single channel properties of either wt or single-point mutants of rENaC. Our data demonstrate that the C-terminal peptides blocked the Liddle's truncation mutant (αβ(T)γ(T)) expressed in Xenopus oocytes but not the single-point mutants (αβ(Y)γ or αβγ(Y)). Moreover, the blocking effect of both peptides in combination on αβ(T)γ(T)-rENaC was synergistic.