We recently demonstrated that endothelial cells are more susceptible than renal tubular epithelial cells to oxidant injury and that renal tubular epithelial cells with proximal tubular characteristics including porcine proximal tubular epithelial cells, opossum kidney proximal tubular epithelial cells, and normal human kidney cortical epithelial cells are more susceptible to oxidant injury than the distal nephron-derived Madin Darby canine kidney cell line. To determine the basis of this differential response, we evaluated several antioxidant defenses in the five cell lines. Glutathione levels were not significantly different among the five cell lines, but catalase and glutathione reductase levels were significantly (p< 0.01) lower in endothelial cells compared to all renal tubular epithelial cells. Among renal tubular epithelial cells, Madin Darby canine kidney cells had significantly (p< 0.05) higher glutathione peroxidase activity. To further evaluate the role of antioxidant defenses in limiting oxidant injury, we determined two responses to oxidant injury (ATP depletion and51Cr release) when glutathione was depleted with buthionine sulfoxamine and when catalase was inhibited with aminotriazole. Oxidant-induced ATP depletion was accentuated when catalase was inhibited as well as when glutathione was depleted with buthionine sulfoxamine. In contrast, inhibition of catalase had little or no effect on51Cr release, whereas glutathione depletion resulted in accentuated51Cr release. We conclude that the increased susceptibility of endothelial cells to oxidant injury as compared with epithelial cells is associated with lower antioxidant defenses. Disruption of the glutathione redox cycle results in accentuated ATP depletion and lytic injury, whereas inhibition of catalase results in accentuated ATP depletion with little effect on lytic injury. Augmented oxidant-induced ATP depletion without augmented cell lysis suggests that ATP depletion alone may not be a critical mediator of cell death in oxidant stress. (Pediatr Res 32: 360–365, 1992)