Alterations in the activity of Cu,Zn-superoxide dismutase (SOD1), an enzyme that converts superoxide (O(.-)(2)) to hydrogen peroxide (H(2)O(2)) plus O(2), have been found to affect cellular susceptibility to oxidative stress and have been invoked as a pathogenetic mechanism in a variety of neurodegenerative diseases. In apparent contradiction, some investigators have found overexpression of SOD1 to be protective whereas others have reported it to be destructive. Furthermore, there has been an ongoing controversy as to whether or not increases in SOD1 activity can in fact lead to an increase in cellular H(2)O(2) levels, one of the mechanisms proposed to explain how SOD1 overexpression may lead to cellular toxicity. Using cells from transgenic mice that express different levels of SOD1, we found that the level of cellular H(2)O(2), determined by fluorescence activated cell sorting in the presence of an H(2)O(2)-sensitive fluorescent dye, increased in parallel with the level of SOD1 activity. Furthermore, we found that this effect was inhibited by overexpression of catalase in these cells, confirming that the increase in fluorescence was indeed due to increases in steady-state H(2)O(2) levels. Increased SOD1 activity was also associated with decreases in cellular O(.-)(2) levels concomitant with the increase in H(2)O(2). Based upon these results, we present a model of cellular susceptibility to oxidative stress as a function of SOD1 activity that suggests a biphasic response. Very low levels of activity can render cells susceptible to oxidative stress because of insufficient metabolism of O(.-)(2). Increasing SOD1 activity from this point is thus expected to be protective. However, as the SOD1 activity increased further, this protective action is lost and actually can lead to cellular injury by overproduction of H(2)O(2), a process that is discussed in terms of recent findings of superoxide reductase activities of this enzyme. This biphasic model may explain how the effects of increases in SOD1 activity depend on the redox state of the cell, and may resolve the apparently paradoxical reports in the literature.