A role of cytoplasmic free calcium [Ca2+]i in hypoxia-induced proximal tubule damage has been proposed. To further investigate the role of [Ca2+]i in mediating hypoxic proximal tubular injury, a video imaging technique has been developed in which [Ca2+]i can be measured simultaneously with propidium iodide (PI) staining of nuclei as an index of hypoxia-induced membrane damage. Hypoxia in rat proximal tubules is associated with a significant rise in [Ca2+]i which precedes evidence of membrane damage as assessed by PI staining. This rise in [Ca2+]i activates calpain, a Ca2+-dependent cysteine protease, and constitutive nitric oxide synthase (NOS), the Ca2+-dependent form of NOS. Inhibition of either calpain or nitric oxide synthase provides marked cytoprotection against hypoxic proximal tubular injury. These observations are consistent with the early rise in [Ca2+]i initiating hypoxic injury by activating NOS and calpain.