In lung epithelial cells, hypoxia decreases the expression and activity of sodium transporting molecules, thereby reducing the rate of transepithelial sodium absorption. The mechanisms underlying the sensing of hypoxia and subsequent coupling to sodium transporting molecules remain unclear. Hydrogen sulfide (H2S) has recently been recognized as a cellular signalling molecule which is enzymatically produced by cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3MST). We have previously shown that H2S-liberating sulphur salts reduce pulmonary transepithelial sodium absorption by inhibition of Na+/K+-ATPase activity (1). Furthermore, airway epithelial cells endogenously produce H2S and H2S production is inversely correlated with O2 concentrations (2). Therefore it was questioned whether endogenously produced H2S contributes to hypoxic inhibition of pulmonary transepithelial sodium absorption. Sodium absorption was measured electrophysiologically in freshly dissected porcine tracheae (obtained from a local slaughterhouse) or cultured human H441 airway epithelial monolayers (as described in (2)) in gas-lift perfusion Ussing chambers. Values are means ± SEM. Hypoxia was established by decreasing O2 concentrations of the Ussing chamber circulation gas, which resulted in an O2 concentration of 2.01 ± 0.04 mg/L (n=3) in the chambers. This hypoxia reversibly decreased normalized amiloride (10 µM)-sensitive sodium current signals from 0.94 ± 0.02 (n=8) to 0.34 ± 0.03 (n=9; p≤0.001; unpaired t-test) in porcine tracheae and from 0.90 ± 0.02 (n=14) to 0.60 ± 0.03(n=9; p≤0.001; unpaired t-test) in H441 monolayers within 30-45 min. The decrease in sodium absorption was due to inhibition of the basolaterally located Na+/K+-ATPase. Normalized Na+/K+-ATPase currents of epithelia which were apically permeabilized with nystatin (75 µM) decreased from 1.49 ± 0.35 (n=7) to 0.64 ± 0.05 (n=8; p ≤0.001; Mann-Whitney U-test) in pig tracheae and from 1.41 ± 0.10 to 0.97 ± 0.16 (n=9; p ≤0.05; unpaired t-test) in H441 monolayers. Pre-treatment (30 min) with the CSE inhibitor D/L-propargylglycine (PAG; 1 mM) decreased normalized hypoxic inhibition of sodium absorption (Ihypoxia) from 0.21 ± 0.02 (n=7) to 0.11 ± 0.03 (n=8; p≤0.01; unpaired t-test) in H441 monolayers, whereas inhibition of CBS (with aminooxy-acetic acid; AOAA; 0.5 mM) or 3MST (with aspartate; 1 mM) had no effect. Inhibition of all of these H2S-generating enzymes with a combination of AOAA, PAG and aspartate decreased Ihypoxia from 0.35 ± 0.03 to 0.03 ± 0.03 (n=6-7; p≤0.001; unpaired t-test) in H441 cells and from 0.56 ± 0.03 to 0.43 ± 0.05 (n=8; p≤0.05; unpaired t-test) in pig tracheae. These data suggest that endogenously produced H2S contributes to hypoxic inhibition of transepithelial sodium absorption in airways.