The regulation of ENaC in the aldosterone sensitive distal nephron is critically important for the body sodium homeostasis and hence for the control of arterial blood pressure. The molecular mechanisms of ENaC regulation are not yet fully understood but probably involve several kinases. In particular aldosterone induced SGK (serum and glucocorticoid inducible kinase) is thought to enhance ENaC surface expression by phosphorylating Nedd4-2 and thereby preventing ENaC retrieval and degradation (Debonneville et al. 2001; Snyder et al. 2002). Recently, we identified an additional regulatory pathway which involves an SGK consensus motif (⁶¹⁶RSRYWS⁶²¹) in the C-terminus of the channel’s α-subunit (Diakov & Korbmacher, 2004). However ⁶¹⁶RSRYWS⁶²¹ sequence is not an exclusive consensus motif for SGK1 but may also be relevant for PKB. PKB is a kinase that is involved in receptor tyrosine kinase regulatory pathways which are activated by insulin and several growth factors. In the present study we investigated the regulation of ENaC activity in outside-out macro patches of Xenopus laevis oocytes heterologously expressing the three subunits (α, β, γ) of rat ENaC. To prevent Na⁺ feedback inhibition the pipette solution had a low Na⁺ concentration containing (mM): 5 NaCl, 90 K-gluconate, 2 EGTA, 10 HEPES, 2 MgATP adjusted to pH 7.3 with Tris. At a holding potential of -70 mV the activity of ENaC was monitored by repeatedly assessing the amiloride (2 μM) sensitive current (ΔI_ami). Current values are given as means ± S.E.M. and significance was evaluated using Student’s paired t test. In control experiments (n=5) ΔI_ami averaged 328 ± 152 pA 5 min after patch excision (ΔI_ami-initial) and remained 24 min after excision at 288 ± 99 pA (ΔI_ami-late). Recombinant constitutively active PKB (T308D, S473D) included in the pipette solution caused a significant (p < 0.05) increase of ENaC currents from 242 ± 82 pA (ΔI_ami-initial) to 905 ± 249 pA (ΔI_ami-late) (n=7). Thus, on average PKB increased ENaC current by about 4-fold. Replacing the serine residue S621 of the ⁶¹⁶RSRYWS⁶²¹ sequence in the C-terminus of the α-subunit by an alanine abolished the stimulatory effect of PKB (n = 6). We conclude that PKB can stimulate ENaC activity, and that this stimulation requires a specific kinase consensus motif in the C-terminus of the channel’s α-subunit. The activation of ENaC by PKB may be relevant for insulin induced stimulation of ENaC in vivo.