The mechanisms of hepatocyte injury caused by exogenous superoxide were investigated with the use of cultured rat hepatocytes. Cell viability, cytosolic free calcium concentration and cell surface structure were observed. Superoxide was produced by adding hypoxanthine and xanthine ozcidase to the buffer. Cytosolic free calcium concentration was calculated by means of ratio imaging of fura 2 fluorescence with multiparameter digitized microscopy. In the buffer containing 1.27 mmol/L of calcium, lactate dehydrogenase release into the buffer began to increase at 1 hr and reached a plateau in 5 hr. Eighteen minutes after the addition of hypoxanthine and xanthine oxidase, small blebs were recognized on the cell surface with a scanning electron microscope; then a gradual rise in cytosolic free calcium concentration was observed. Thirty minutes after exposure to superoxide, large blebs were recognized with a phase-contrast microscope, when cytosolic free calcium concentration had risen to about 700 nmolL. Depriving the buffer of calcium ( < 10 pmolL) significantly suppressed bleb formation and cell death, and cytosolic free calcium concentration was found to remain around the basal level (200 nmol/L). When ethylene glycol-bie (@-aminoethyl ether)-N,N,N',N'-tetraacetic acid was added to the buffer, bleb formation and cell death were suppressed more completely, and cytosolic free calcium concentration decreased. Superoxide dismutase combined with catalase or nifedipine allowed the hepatocytes to maintain their viability and suppressed cytosolic free calcium concentration elevation. Calpeptin, a Ca2 + -dependent neutral protease inhibitor, did not affect the rise in cytosolic free calcium concentration but prevented cell injury. We concluded that cell injury caused by superoxide is initiated by influx of calcium and that the resultant activation of Caz+-dependent protease may play an important role in bleb formation and cell death. (HEPATOLOGY 1994;191223-1228.) Calcium has been suggested to play a crucial role in the course of cell injury caused by ischemia and