Acute hypoxia is well known to regulate [Ca²⁺]_i indirectly by altering the activity of plasmalemmal ion channels (e.g. Peers & Kemp, 2001). However, its direct effects on intracellular Ca²⁺ homeostasis are less well studied. Here, we have investigated the effects of acute hypoxia on agonist-evoked changes of [Ca²⁺]_i in human saphenous vein endothelial cells. Cells were isolated and maintained in primary culture as previously described (Budd et al., 1991) and [Ca²⁺]_i was monitored in Fura-2 loaded cells as previously described (Smith et al., 2003). Bath application of 10μM ATP in the absence of external Ca²⁺ evoked a transient rise in cytosolic [Ca²⁺] (peak = 0.211 ±0.008 ratio units (ru; n = 118 cells). Exposure of cells to hypoxia (pO2 ~ 25mmHg) for 150s caused a small but distinct increase in [Ca²⁺]_i (peak 0.012 ± 0.002 ru; n = 46). Following this exposure, subsequent application of 10μM ATP evoked a transient rise of [Ca²⁺]_i (peak, 0.134 ± 0.007 ru; n=37) which was significantly smaller than that observed in cells not exposed to hypoxia (p<0.001, unpaired Student's t-test). Following exposure to either ATP or to acute hypoxia, re-introduction of Ca²⁺ to the perfusate evoked a similar degree of capacitative Ca²⁺ entry in both cases, peaking at 0.080 ± 0.006 ru following ATP exposure and 0.077 ± 0.009 ru following exposure to hypoxia (n=36 in each case). Our data indicate that acute hypoxia evokes Ca²⁺ release from an ATP-sensitive intracellular pool in human venous endothelial cells, and such release is sufficient to trigger capacitative Ca²⁺ entry.