When hypertonicity is imposed with sufficient intensity and acuteness, cells die. Here we investigated cellular pathways involved in the death using cell lines derived from renal epithelia. We found that hypertoncity rapidly induced activation of intrinsic cell death pathway - release of cytochrome c and activation of caspase-3 and caspase-9 - and extrinsic pathway - activation of caspase-8. Likewise, a lysosomal pathway of cell death characterized by partial lysosomal rupture and release of cathepsin B from lysosomes to the cytosol was also activated. Relationship among the pathways was examined using specific inhibitors. Caspase inhibitors did not affect cathepsin B release into the cytosol by hypertonicity. In addition, cathepsin B inhibitors and caspase inhibitors did not affect hypertoncity-induced cytochrome c release suggesting that the three pathways were independently activated. Combined inhibition of caspases and cathepsin B conferred significantly more protection from hypertonicity-induced cell death than inhibition of caspase or cathepsin B alone indicating that all the three pathways contributed to the hypertonicity-induced cell death. Similar pattern of sensitivity to the inhibitors was observed in two other cell lines derived from renal epithelia. We conclude that multiple cell death pathways are independently activated early in response to lethal hypertonic stress in renal epithelial cells.