The serum- and glucocorticoid-inducible kinase 1 (SGK1) has been identified as a signalling molecule up-regulated by aldosterone, which stimulates the renal epithelial Na(+) channel ENaC. It is therefore thought to participate in the antinatriuretic action of this hormone. More recently, two isoforms, SGK2 and SGK3, have been cloned. The present study was performed to establish whether SGK2 and SGK3 influence ENaC activity similarly to SGK1. Dual-electrode voltage-clamp experiments in Xenopus laevis oocytes expressing alpha,ss,gamma-ENaC with or without SGK1, SGK2 or SGK3 revealed a stimulatory effect of all three kinases on the amiloride-sensitive current (I(Na)). To establish whether the SGK isoforms exert their effects through direct phosphorylation, we replaced the serine at the SGK consensus site of alphaENaC (alpha(S622A)ENaC) by site-directed mutagenesis. alpha(S622A),beta,gamma-ENaC was up-regulated similar to wild-type ENaC, suggesting that SGK isoforms do not act via direct phosphorylation of the transport proteins. In conclusion, SGK2 and SGK3 mimic the function of SGK1 and are likely to participate in the regulation of ENaC activity.