The epithelial sodium channel is a multimeric protein formed by three homologous subunits: α, β, and γ; each subunit contains only two transmembrane domains. The level of expression of each of the subunits is markedly different in various Na+ absorbing epithelia raising the possibility that channels with different subunit composition can function in vivo. We have examined the functional properties of channels formed by the association of α with β and of α with γ in the Xenopus oocyte expression system using two-microelectrode voltage clamp and patch-clamp techniques. We found that αβ channels differ from αγ channels in the following functional properties: (a) αβ channels expressed larger Na+ than Li+ currents (I(Na+)/I(Li+) 1.2) whereas αγ channels expressed smaller Na+ than Li+ currents (I(Na+)/I(Li+) 0.55); (b) the Michaelis Menten constants (K(m)) of activation of current by increasing concentrations of external Na+ and Li+ of αβ channels were larger (K(m) > 180 mM) than those of αγ channels (K(m) of 35 and 50 mM, respectively); (c) single channel conductances of αβ channels (5.1 pS for Na+ and 4.2 pS for Li+) were smaller than those of αγ channels (6.5 pS for Na+ and 10.8 pS for Li+); (d) the half-inhibition constant (K(i)) of amiloride was 20-fold larger for αβ channels than for αγ channels whereas the K(i) of guanidinium was equal for both αβ and αγ. To identify the domains in the channel subunits involved in amiloride binding, we constructed several chimeras that contained the amino terminus of the γ subunit and the carboxy terminus of the β subunit. A stretch of 15 amino acids, immediately before the second transmembrane domain of the β subunit, was identified as the domain conferring lower amiloride affinity to the αβ channels. We provide evidence for the existence of two distinct binding sites for the amiloride molecule: one for the guanidium moiety and another for the pyrazine ring. At least two subunits α with β or γ contribute to these binding sites. Finally, we show that the most likely stoichiometry of αβ and αγ channels is 1α:1β and 1α:1γ, respectively.