The mechanisms underlying the hormonal regulation of human airway epithelial Na⁺ and Cl^– conductance (G_Na and G_Cl respectively) were explored by recording membrane currents from H441 cells using the perforated patch recording configuration (see Clunes et al., 2004). Studies of cells maintained in hormone-free medium (prepared using dialysed serum), showed that the resting membrane potential (V_m) was essentially identical to E_K (-82 mV) and was unaffected by reducing [Na⁺]_o to 10 mM (NMDG⁺ replacement; control: -83.8 ± 7.1 mV; low Na⁺: -91.4 ± 6.6 mV, n = 5. mean ± s.e.m.) or lowering [Cl^–]_o to 29 mM (gluconate replacement; control: -79.9 ± 2.3 mV; low Cl^–: -78.9 ± 2.4 mV; n = 5). Raising [K⁺]_o to 134.5 mM (Na⁺ substitution) caused depolarization (control: -80.1 ± 4.3 mV; high K⁺: -21.8 ± 2.3 mV; n = 8; P < 0.001; Student’s paired t test). G_Na and G_Cl are therefore negligible under these conditions and the electrical properties of hormone-deprived H441 cells are dominated by G_K. Studies of cells maintained (~24 h) in medium supplemented with dexamethasone (0.2 µM) or insulin (20 nM) showed that both hormones depolarized V_m by ~20 mV (P < 0.001 for both). As anticipated (Clunes et al. 2004), lowering [Na⁺]_o to 10 mM hyperpolarized dexamethasone treated cells (control: -60.0 ± 2.4 mV; low Na⁺: -106.2 ± 6.4 mV) confirming that this glucocorticoid depolarizes V_m by increasing G_Na. Lowering [Cl^–]_o had no effect V_m (control: -65.9 ± 3.8 mV; low Cl^–: -58.0 ± 6.1 mV; n = 4) indicating that dexamethasone does not control G_Cl. However, lowering [Na⁺]_o had no effect upon insulin-treated cells (control: 63.3 ± 5.0 mV; low Na⁺: -62.4 ± 4.5 mV) whilst reducing [Cl^–]_o caused further depolarization (control: -57.6 ± 2.2 mV; low Cl^–: -39.9 ± 2.5 mV; n = 5; P < 0.001); the depolarizing response to insulin thus reflects an increase in G_Cl. Subsequent studies showed that the response to dexamethasone was mimicked by transient expression of constituitively active serum and glucocorticoid-regulated kinase 1 (SGK1) whilst the response to insulin was reproduced by an active form of the phosphatidylinositol trisphosphate-dependent kinase (PI3K). Insulin and dexamethasone thus appear to depolarize H441 cells by evoking SGK1-dependent control over G_Na and and PI3K-dependent control over G_Cl respectively. Moreover, although PI3K-dependent control of ENaC is well documented (e.g. Tong et al., 2004) the present data indicate that, at least in hormone-deprived H441 cells, SGK1 can increase G_Cl without affecting G_Na.