Smooth muscle cells were obtained by explant culture from segments of human internal mammary artery taken (with approval from the local research ethics committee and informed patient consent) from patients undergoing routine coronary bypass surgery. Cells were cultured at 37°C in a humidified atmosphere with 5% CO₂ in air. Experiments were conducted at passage 1. Chronic hypoxia was imposed 24 hours prior to experiments by switching cells to a gas mixture of 2.5% O₂, 5% CO₂ and balance N₂. Intracellular Ca²⁺ ([Ca²⁺]_i) was monitored in fura-2-loaded cells as previously described (Smith et al. 2003). Results are given as % changes in the 340/380 nm fluorescence ratio. Values have been determined from measurements in at least 7 cells (and as many as 90) from between 3 and 12 patients. Data have been analysed by Student’s unpaired t test or one-way ANOVA as appropriate. The basal [Ca²⁺]_i measured (as the 340/380 nm fluorescence ratio) in human internal mammary artery smooth muscle cells (IMASMC) bathed in Ca²⁺-containing (2.5mM) solution was not significantly altered by chronic hypoxia. However, on removal of external Ca²⁺ (solution contained 0mM CaCl₂ and 1mM EGTA), a notable drop in [Ca²⁺] was observed in the normoxic cells which was not seen in cells exposed to chronic hypoxia. Perfusion of normoxic cells with a high K⁺ (50mM) solution caused a rapid, sustained and reversible rise in [Ca²⁺]_i. In cells exposed to chronic hypoxia this high K⁺-induced Ca²⁺ elevation was significantly reduced by 38% (compared to the normoxic cells; P<0.01). In both normoxic and hypoxic cells the high K⁺-induced increase in [Ca²⁺]_i was abolished by removal of external Ca²⁺ or by blockade of both L-type and T-type voltage-operated Ca²⁺ channels (VOCC) with 2 μM nifedipine and 3 μM pimozide, respectively (P<0.01 for each condition). Use of nifedipine alone inhibited the K⁺-induced rise in [Ca²⁺]_i by approximately 90% in normoxic cells (P<0.01) and 72% in hypoxic cells (P<0.01), while pimozide alone inhibited the response by approximately 27% in normoxic cells (although this decrease was not significant) and 47% in hypoxic cells (P<0.01). These data indicate that in proliferative human IMASMC, chronic hypoxia leads to changes in Ca²⁺ handling compared to normoxic controls. The lack of effect of removing external Ca²⁺ on the basal [Ca²⁺]_i of hypoxic cells is consistent with an inhibition of Ca²⁺ extrusion mechanisms under hypoxic conditions, although further experiments are required to confirm this. In addition, chronic hypoxia was found to decrease the size of the high K⁺-induced increase in [Ca²⁺]_i, consistent with hypoxic modulation of VOCC in these cells.