Radicals including superoxide anions, hydrogen peroxide or hydroxyl radicals and NO or peroxynitrite cause the breakage of DNA strands and activation of poly–(ADP–ribose) synthase (PARS). Recent studies showed that inhibition of PARS activity reduces the tissue injury after exposure to oxidative stress. However, the role of PARS in renal injury by oxidants has not been examined. In this study effect of a PARS inhibitor, 3–aminobenamide (3–AB), on injury of opossum kidney or LLC–PK<sub>1</sub> cells by hydrogen peroxide or tert–butyl hydroperoxide (t–BHP) was examined. The exposure of opossum kidney cells to hydrogen peroxide activated PARS and decreased cellular adenosine triphosphate levels in a concentration–dependent manner. Inhibition of PARS with 3–AB prevented the cell death induced by hydrogen peroxide and also prevented adenosine triphosphate depletion. 3–AB did not have hydroxyl radical scavenging effect. In contrast, t–BHP did not affect the PARS activity. The decrease in cellular adenosine triphosphate levels by t–BHP was less than that by hydrogen peroxide. 3–AB failed to prevent the cell death induced by t–BHP. PARS activation after exposure of hydrogen peroxide was inhibited by addition of t–BHP. However, t–BHP showed an additive effect on cell death with hydrogen peroxide. These results indicate that activation of PARS plays an important role in hydrogen peroxide induced injury in opossum kidney cells and that hydrogen peroxide and t–BHP induce cell injury by different mechanisms.