Serum and glucocorticoid inducible kinase 1 (SGK1) is a key regulator of the epithelial sodium channel (ENaC) in its αβγ subunit configuration. We demonstrated that in rat ENaC the serine residue 621 (S621) localised in an SGK consensus motif (RXRXX(S/T)) in the channel’s α-subunit is critically important for acute channel activation by SGK1 and the kinases PKBα and DYRK2 (1, 2, 3). Phosphorylation at S621 probably affects ENaC gating and turns previously silent channels into channels with a high open probability. Interestingly, this is reminiscent of proteolytic ENaC activation observed in single-channel recordings from outside-out patches exposed to trypsin or chymotrypsin (4, 5). Proteolytic channel activation is a unique feature of ENaC, but the underlying molecular mechanisms and a possible interplay with channel regulation by phosphorylation remain incompletely understood. The aim of the present study was to explore whether human ENaC can also be activated acutely by SGK1 and whether this activation depends on the highly conserved serine residue S594 in human αENaC.  In addition, we investigated whether ENaC activantion by SGK1 depends on the cleavage state of γENaC. Human αβγENaC and mutant ENaC constructs were heterologously expressed in Xenopus laevis oocytes. Amiloride (2 µM) was applied to determine amiloride-sensitive ENaC currents (ΔI_ami) in outside-out macro-patches. We demonstrated that in outside-out patches from oocytes expressing human ENaC recombinant and constitutively active SGK1 included in the pipette solution increased ΔI_ami from 69 ± 21 pA to 141 ± 27 pA (n=8; SEM; p<0.003, paired t-test) within ~24 min after patch excision. Importantly, this stimulatory effect was completely abolished when the conserved serine residue S594 was replaced by an alanine in the channel’s α-subunit by site-directed mutagenesis (n=8). It is well established that during ENaC maturation proteolytic cleavage occurs at three putative furin sites (two in α- and one in γENaC) before the channel reaches the plasma membrane. The final critical step in proteolytic ENaC activation probably takes place at the plasma membrane where γENaC is cleaved by membrane bound proteases and/or extracellular proteases in a region distal to the furin site. By Western blot analysis we confirmed for human γENaC the previously reported finding (4, 5) that in the oocyte expression system γ-ENaC is fully cleaved at its furin cleavage site by endogenous convertases. Interestingly, replacing in human γENaC all four residues of the putative furin cleavage site by alanines (135AAAA138) completely abolished the stimulatory effect of SGK1 (n=10). In contrast, the stimulatory effect of the small ENaC activator S3969 (10 μM) was preserved indicating that the mutant channel is functional and can be activated by an SGK1 independent mechanism. In conclusion, our findings highlight the importance of the conserved serine residue S594 in human αENaC for mediating the acute channel activation by SGK1. Moreover, our data indicate that ENaC activation by SGK1 requires prior cleavage of γENaC at its furin cleavage site. Thus, these findings suggest an interdependence of proteolytic channel processing and acute channel activation by SGK1.