Aminotriazole-mediated inhibition of catalase has been used in previous studies as a measure of in vivo changes in the hydrogen peroxide generation. Using this method, we found a significantly higher inhibition of renal catalase activity at various time points (30, 60, and 90 min) in glycerol-treated rats (a well-established model for myoglobinuric acute renal failure) compared with rats treated with aminotriazole alone. The greater inhibition in the glycerol-treated group was not due to differences in aminotriazole levels. We confirmed that catalase inactivation by aminotriazole was due to formation of catalase-hydrogen peroxide intermediate (compound I) because catalase inactivation was prevented by ethanol, a competitive substrate for compound I. There were no significant differences in the aminotriazole-induced inhibition of renal cortical catalase activity in control and uranyl nitrate-treated rats, suggesting that there was no enhanced generation of hydrogen peroxide in this model of acute renal failure. Taken together, these data provide evidence for enhanced generation of hydrogen peroxide in glycerol-induced acute renal failure and suggest that the enhanced generation of hydrogen peroxide in the glycerol-induced acute renal failure is not a result of nonspecific response to tissue injury.