Attenuated vasoconstrictor reactivity following chronic hypoxia (CH) is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization and diminished intracellular [Ca²⁺]. We tested the hypothesis that increased production of nitric oxide (NO) after CH contributes to blunted vasoconstrictor responsiveness. We found that basal NO production of mesenteric arteries from CH rats (barometric pressure = 380 Torr; 48 h) was greater than that of controls (barometric pressure = 630 Torr). In addition, studies employing pressurized mesenteric arteries (100–200 μM ID) abluminally loaded with the Ca²⁺ indicator fura 2-AM demonstrated that although NO synthase (NOS) inhibition normalized agonist-induced vasoconstrictor responses between groups, VSM cell [Ca²⁺] in vessels from CH rats remained diminished compared with controls. To determine whether elevated NO production following CH results from increased NOS protein levels, we performed Western blots for NOS isoforms by using mesenteric arteries from control and CH rats. Endothelial NOS levels did not differ between groups, and other NOS isoforms were not detected in these samples. Selective endothelial loading of fura 2-AM was employed to test the hypothesis that elevated endothelial cell [Ca²⁺] following CH accounts for enhanced NOS activity. These experiments demonstrated greater endothelial cell [Ca²⁺] in mesenteric arteries isolated from CH rats compared with controls. We conclude that enhanced production of NO resulting from elevated endothelial cell [Ca²⁺] contributes to attenuated reactivity following CH by decreasing VSM cell Ca²⁺sensitivity.