We have previously identified three homologous subunits alpha, beta, and gamma of the highly selective amiloride-sensitive Na channel from the Xenopus laevis kidney A6 cell line, which forms a tight epithelium in culture. We report here two novel genes, termed beta2 and gamma2, which share 90 and 92% sequence identity with the previously characterized beta and gamma XENaC, respectively. beta2 and gamma2 transcripts were detected in lung, kidney, and A6 cells grown on porous substrate. The physiological and pharmacological profile of the Na channel expressed after alphabeta2gamma XENaC cRNA injection in Xenopus oocyte did not differ from alphabetagamma XENaC. By contrast, the channel expressed after alphabetagamma2 injection showed: (i) a lower maximal amiloride-sensitive sodium current, (ii) a higher apparent affinity for external sodium and inactivation of the sodium current by high sodium concentrations, and (iii) a lower apparent affinity for amiloride (KI alphabetagamma2; 1.34 microM versus alphabetagamma 0.35 microM). These data indicate that the gamma (and/or gamma2) subunit participates in amiloride binding and the sensing of the extracellular sodium concentration. The close homology between gamma and gamma2 will help to define the domains involved in sensing external sodium and in the structure of this important drug receptor.