The effect of chronic hypoxia on free intracellular Ca2+ concentration ([Ca2+]i) and Ca2+ sensitivity of myofilaments during agonist stimulation was examined in uterine arteries obtained from normoxic and chronically hypoxic pregnant sheep maintained at high altitude (3,820 m) for approximately 110 days. Smooth muscle [Ca2+]i was measured simultaneously with muscle contraction in the same intact tissue. Whereas both KCl and 5-HT increased [Ca2+]i and tension simultaneously in the uterine artery, 5-HT produced significantly greater contractile tension (in g) than KCl at a given amount of [Ca2+]i as indicated by the ratio of fura 2 fluorescence intensity induced by excitation at 340 nm to that induced at 380 nm (29.8 +/- 6.9 vs. 16.9 +/- 4.0, P < 0.05). Chronic hypoxia did not change KCl-induced contractions, nor did it affect KCl-mediated increases in [Ca2+]i. In contrast, chronic hypoxia significantly inhibited 5-HT-induced contractions and decreased the 5-HT-stimulated increase in [Ca2+]i (pD2 7.46 +/- 0.18-->6.86 +/- 0.11, P < 0.05, where pD2 is -log half-maximal effective concentration) in uterine arteries. In addition, the slope (g tension/nM [Ca2+]i) of the 5-HT-mediated [Ca2+]i-tension relationship was significantly decreased in chronically hypoxic arteries (0.024 +/- 0.002-->0.013 +/- 0.001, P < 0.01). The results suggest that chronic hypoxia suppresses agonist-mediated Ca2+ homeostasis in uterine arteries by inhibiting Ca2+ mobilization and the agonist-enhanced Ca2+ sensitivity of myofilaments.