Epithelial Na+ channel (ENaC) activity, which determines the rate of renal Na⁺ reabsorption, can be regulated by G-protein coupled receptors. Regulation of ENaC by Gα mediated downstream effectors has been studied extensively, but the effect of Gβ dimers on ENaC is unclear. A6 cells endogenously contain high levels of Gβ1 but low levels of Gβ3 ,Gβ4 and Gβ5 were detected by Q-PCR. We tested G₂ combined individually with G_β1 through G_β5 expressed in A6 cells, after which we recorded single ENaC activity. Among the five β and 2 combinations, β12 strongly inhibits ENaC activity by reducing both ENaC channel number (N) and open probability (Po) compared to control cells. In contrast, the other four β isoforms combined with 2 have no significant effect on ENaC activity. By using various inhibitors to probe G_β12 effects on ENaC regulation, we found that G_β12-mediated ENaC inhibition involved activation of phospholipase C-β and its enzymatic products that induce protein kinase C and ERK1/2 signaling pathways.