The regulation of the epithelial sodium channel (ENaC) is thought to involve phosphorylation and dephosphorylation events. Recently we demonstrated that S621 in a putative SGK consensus motif (RXRXX(S/T)) of rat aENaC is critically important for channel activation by recombinant SGK1 applied to the cytosolic surface of outside-out patches (Diakov & Korbmacher 2004, J. Biol. Chem. 279:38134–42). However, in vitro studies suggest that S621 is unlikely to be phosphorylated by SGK1 since it is followed by a highly conserved proline residue (Alessi et al. 1996, FEBS Lett. 399:333–8). In contrast, S621 is a potential phosphorylation site for DYRK2 and its phosphorylation may prime the preceding S617 residue to be phosphorylated by GSK3. Therefore, we investigated the regulation of ENaC by these two kinases in outside-out macropatches of X. laevis oocytes expressing rat ENaC. Within 24 min after patch excision recombinant DYRK2 in the pipette solution increased ENaC currents to 195 ± 26% (n=6; ± SEM; p<0.01) of its starting value while GSK3 decreased ENaC currents to 29 ± 5% (n=10; p<0.001). Replacing serine S621 by alanine abolished the effects of both kinases. We conclude that SGK1 may modulate ENaC activity in vivo by affecting the complex and sequential interplay of other kinases, e.g. DYRK2 and GSK3.