To investigate the effect of extracellular pH on endothelial nitric oxide synthase (eNOS) in human pulmonary arteries, we measured eNOS activity and expression as well as some ion channels in human pulmonary arterial endothelial cells (HPAEC) exposed to various pH levels (6.6–8.0). eNOS activity was found to increase with alkalization and decrease with acidification, while Ca²⁺ uptake into HPAEC increased with alkalization. The addition of 3′,4′-dichlorobenzamil hydrochloride, an inhibitor of the Na⁺/Ca²⁺ exchanger (NCX), prevented the increase of eNOS activity with alkalosis. Exposure to alkalosis and acidosis increased eNOS and NCX mRNA levels. These results suggest that an elevation of extracellular pH activates eNOS via the influx of extracellular Ca²⁺ and that NCX also regulates eNOS activity during alkalosis. Furthermore, NCX may have a tight interaction with eNOS at the level of transcription and might affect pulmonary circulation during alkalosis and acidosis.