Objective: The epithelial sodium channel (ENaC) is the rate limiting step for sodium absorption in the aldosterone sensitive distal nephron. In outside-out patches ENaC activation by SGK1 critically depends on the serine residue 621 (S621) in the a-subunit of rat ENaC (Diakov & Korbmacher, J Biol Chem 2004, 279: 38134). Phosphorylation of S621 may prime the preceding S617 for phosphorylation by GSK3. The aim of the present study was to investigate the effect of GSK3 on ENaC activity. Methods. Patch clamp studies were performed on microdissected mouse cortical collecting ducts (CCD) and on Xenopus laevis oocytes heterologously expressing wild-type rat ENaC or channels with mutations in the a-subunit (S621A or S617A). ENaC activity was determined by measuring the amiloride-sensitive current (DI_Ami). Pipette solution contained active or heat-inactivated recombinant GSK3. Results. GSK3 in the pipette solution did not affect whole-cell DI_Ami in mouse CCD cells. However, in excised outside-out patches initial DI_Ami averaged 2.8±0.6% of the corresponding whole-cell DI_Ami with heat-inactivated GSK3 in the pipette solution but only 1.2±0.5% with active GSK3. Moreover, within 10 min after patch excision DI_Ami declined to 34±9% (p<0.05) of its initial value when GSK3 was present. In contrast, in control experiments with heat-inactivated GSK3 DI_Ami remained at 61±9% of its initial value. A similar inhibitory effect of active GSK3 on ENaC activity was observed in outside-out patches of oocytes. Importantly, the S617A and S621A mutations prevented the inhibitory action of GSK3. Conclusion. Our data indicate that GSK3 inhibits ENaC activity in native renal CCD. The inhibitory effect of GSK3 probably involves phosphorylation of S617 in the a-subunit following a priming phosphorylation of S621. Thus, ENaC inhibition by GSK3 may serve as a negative feedback mechanism.