Acute hypoxic inhibition of potassium (K+) channels is a critical step in regulatory processes designed to link lowering of oxygen levels to cellular responses. In the pulmonary circulation the vascular tone in response to hypoxia is determined in part by oxygen-sensitive ion channels but the molecular mechanism behind is still unknown. Methods: Primary smooth muscle cells were isolated from human pulmonary arteries (hPASMC) from patients (n = 20) undergoing lung surgery for lung cancer without a history of pulmonary vascular disease or arterial hypoxemia as described earlier (Olschewski et al 2006). The study protocol for tissue donation was approved by the “Institutional Review Board” of the Medical University of Graz in accordance with national law and with guidelines on Good Clinical Practice/International Conference on Harmonization. Written informed consent was obtained from each individual patient. Hypoxia-induced changes in ion conductance and in intracellular calcium homeostasis of hPASMC were investigated by switching the perfusing medium from normoxia to moderate hypoxia (pO2 of 25-35 mmHg). The whole-cell patch-clamp technique was used as previously described (1). Changes in intracellular calcium were measured by using fura-2am loaded hPASMC. Immunofluorescence of phosphorylated tyrosine kinase in normoxia and after 30 minutes of hypoxia was performed using phosphospecfic antibody. Intergroup differences were assessed by a factorial analysis of variance with post hoc analysis with Fisher’s least significant difference test. Results: Voltage-activated (Kv), calcium-sensitive (KCa) and non-inactivating TASK-1 K+ currents were reversibly inhibited by moderate hypoxia. The reduction of Kv current measured at +50 mV was 43±3% (n=8, p<0.01) and 56±5% for KCa (n=7, p<0.01). TASK-1 current was blocked to 51±3% (n = 22, p<0.001) at 0 mV. PP2, a selective inhibitor of Src thyrosine kinases abolished the effect of hypoxia (n=25), whereas PP3 (n=16), Ro-31-8220 and Gö 6983 (PKC inhibitors, n=18), KT 5720 (PKA inhibitor, n=15), Y-27632 (Rho-kinase inihibitor, n=16) or compound C (AMP kinase inhibitor, n=20) failed to attenuate the hypoxia-induced reduction of the currents. Hypoxic challenges induced a transient increase in intracellular calcium concentration (n=50). The effect of hypoxia was abolished when hPASCM were preincubated with PP2 (n=40). In hPASMC a redistribution of phosphorylated tyrosine kinase fluorescence to the plasma membrane was observed under hypoxic conditions, whereas it was localized in the cytosol in normoxic cells (n=3). Conclusion: Our data indicate that the thyrosine kinase (src) pathway is required for the acute response to hypoxia of K+ channels and intracellular calcium homeostasis in primary human PASMC.