The present study addressed whether chronic hypoxia is associated with reduced nitric oxide (NO) release due to decreased activation of endothelial NO synthase (eNOS). Primary cultures of endothelial cells from human umbilical veins (HUVECs) were used and exposed to different oxygen levels for 24 hours, after which NO release, intracellular calcium, and eNOS activity and phosphorylation was measured after 24 hours. Direct measurements using a NO microsensor showed that in contrast to 1 hour exposure to 5% and 1% oxygen (acute hypoxia), histamine-evoked (10 µM) NO release from endothelial cells exposed to 5% and 1% oxygen for 24 hours (chronic hypoxia) was reduced by, respectively, 58% and 40%. Furthermore, chronic hypoxia also lowered the amount and the activity of eNOS enzyme. The decrease in activity could be accounted for by reduced intracellular calcium and altered eNOS phosphorylation. eNOS-Ser¹¹⁷⁷ and eNOS-Thr⁴⁹⁵ phosphorylations were reduced and increased, respectively, consistent with lowered enzyme activity. The Akt kinase, which can phosphorylate eNOS-Ser¹¹⁷⁷, was also decreased by hypoxia, regarding both total protein content and the phosphorylated (active) form. Moreover the protein content of -actin, which is known to influence the activity of eNOS, was almost halved by hypoxia, further supporting the fall in eNOS activity. In conclusion, chronic hypoxia in HUVECs reduces histamine-induced NO release as well as eNOS expression and activity. The decreased activity is most likely due to changed eNOS phosphorylation, which is supported by decreases in Akt expression and phosphorylation. By reducing NO chronic hypoxia may accentuate endothelial dysfunction in cardiovascular disease.