The aim of this study was to assess whether hypoxia inhibits endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production, and whether iptakalim may rescue human pulmonary artery endothelial cells (HPAECs) from hypoxia-induced NO system dysfunction. HPAECs were cultured under hypoxic conditions in the absence or presence of 0.1, 10 and 1,000 μM iptakalim or the combination of 10 μM iptakalim and 1, 10 and 100 μM glibenclamide for 24 h, and the eNOS activity and NO levels were measured in the conditioned medium from the HPAEC cultures. The eNOS activity and NO levels were reduced significantly in the conditioned medium from HPAEC cultures under hypoxic conditions. Pre-treatment with 10 μM iptakalim normalized the reduction of the eNOS activity and NO levels caused by hypoxia in the conditioned medium from HPAEC cultures. Iptakalim raised the eNOS activity and NO levels under hypoxic conditions, but was blocked by the K(ATP) channel blocker, glibenclamide. Our results indicate that hypoxia impairs NO system function, whereas the ATP-sensitive K(+) channel opener, iptakalim, may rescue HPAECs from hypoxia-induced NO system dysfunction.