We propose that generation of reactive oxygen species (ROS) during ischemia is associated with an increase in intracellular calcium ([Ca2+]i) in pulmonary capillary endothelial cells. We used an isolated rat lung model and epifluorescence microscopy to evaluate [Ca2+]i in subpleural microvascular endothelial cells in situ by ratio imaging of the fluorophores, Calcium Green and Fura Red (CG/FR). Lungs were ventilated continuously under control (continuously perfused) or global ischemia (no perfusion) and thus remained adequately oxygenated even with ischemia. Ischemia for 5 min led to increase in CG/FR, indicating increase in [Ca2+]i in endothelial cells in situ; CG/FR remained elevated during a subsequent 10 min of ischemia. Ca(2+)-free perfusion and gadolinium (100 microM) inhibited the increase in [Ca2+]i, while thapsigargin (250 nM) had no effect. These results indicate that increase in endothelial cell [Ca2+]i with ischemia was due to influx from the extracellular medium. Perfusion with N-acetyl-L-cysteine (20 mM) or diphenyleneiodonium chloride (10 microM) prevented the ischemia-mediated [Ca2+]i increase, suggesting a role for ROS in the Ca2+ changes with ischemia. Membrane depolarization by perfusion with high potassium (K+) or glyburide also resulted in increased [Ca2+]i whereas the K(+)-channel agonist cromakalim, inhibited ischemia-mediated Ca2+ influx. We conclude that increased ROS generation with 'oxygenated' lung ischemia is associated with influx of Ca2+ and an increase in endothelial cell cytosolic calcium concentration.