Blood oxygen tension (PO₂) is an important local factor in the regulation of circulation. Arteries and arterioles are able to sense changes in PO₂ in the blood or surrounding tissues and acutely respond with a vasomotor response (constriction or dilation) that depends on the vascular territory. Resistance pulmonary arteries constrict in response to a decrease in alveolar PO₂ (alveolar hypoxia), thereby diverting blood flow from poorly ventilated lung regions towards areas with a higher PO₂, optimizing the ventilation/perfusion ratio. Under conditions of persistent hypoxemia (low blood PO₂), hypoxic pulmonary vasoconstriction (HPV) contributes to the pathogenesis of pulmonary hypertension. Unlike resistance pulmonary arteries, conduit pulmonary vessels and most systemic arteries dilate in response to hypoxia. Hypoxic vasodilation (HVD) also has a relevant physiologic role as it favors the perfusion of O₂-deprived tissues. Previous findings have suggested that voltage-gated L-type Ca²⁺ channels in vascular smooth muscle are O₂-sensitive and may therefore critically contribute to acute HVD and HPV. However, how variations of O₂ tension are detected and the mechanisms whereby these changes are conveyed to membrane ion channels have remained elusive. We have observed: i) an antagonistic modulation of Ca²⁺ channel activity in systemic (inhibition) vs pulmonary resistance (potentiation) myocytes, under acute hypoxia, accompanied by corresponding changes in intracellular calcium concentration; ii) a direct modulation of L-type Ca²⁺ channel currents by NADH and H₂O₂ in these cells; and iii), that the effect of hypoxia on Ca²⁺ currents and cytosolic Ca²⁺ levels is dependent on mitochondrial complex I (MCI) activity, as it is absent in myocytes from genetically modified MCI-SM mice (lacking the NDUFS2 subunit, a component of MCI catalytic core). These findings suggest that MCI plays a key role in acute O₂ sensing by myocytes from systemic and pulmonary resistance arteries, likely by mediating the production of signaling molecules (NADH and H₂O₂) that regulate membrane Ca²⁺ channels. Furthermore, mice exposed to chronic hypoxia (10% O₂ tension for 3-4 weeks) exhibit increases in medial wall thickness of pulmonary resistance arteries as well as the right ventricular area. The changes induced by chronic hypoxia were abolished or markedly reduced in MCI-SM mice. Overall, these results strongly suggest that mitochondria may not only play an essential role in acute vascular O₂ sensing and signaling, but also in the (mal)adaptive responses of pulmonary vessels to chronic hypoxia.