The role of different antioxidant pathways in cultured rat pleural mesothelial cells was studied by exposing the cells to various hydrogen peroxide (H2O2) concentrations and by measuring H2O2 cell cytotoxicity and the capacity of the cells to scavenge H2O2. The antioxidant enzymes, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and catalase were analyzed biochemically. Catalase and CuZn superoxide dismutase were localized by immunocytochemistry. To enable investigation of the glutathione redox cycle and catalase pathways, glutathione reductase was inactivated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and catalase was inactivated with aminotriazole. When the cells were exposed to a low, sublethal (0.030 mM) H2O2 concentration, glutathione reductase but not catalase inactivation resulted in a decreased capacity to remove H2O2 from the extracellular medium. When the cells were exposed to a high (0.25 mM) H2O2 concentration, H2O2-scavenging capacity decreased remarkably when catalase was inactivated. When the cells were exposed to 0.1 to 0.5 mM H2O2, cell cytotoxicity (lactate dehydrogenase release) increased significantly if glutathione reductase was inactivated; catalase inactivation resulted in a significant cytotoxicity only at high (greater than or equal to 0.25 mM) H2O2 concentrations. Immunocytochemical studies showed that the cells, both in situ and in vitro, contained low amounts of catalase. This suggests that the results of the catalase-inhibition studies are probably not due to a change in the characteristics of the cells in culture. 3-Aminobenzamide is a compound that is known to prevent NAD depletion through inhibition of poly(ADP-ribose) polymerase during oxidant stress. When intact cells were treated with different antioxidants and exposed to 0.5 mM H2O2, both catalase and 3-aminobenzamide protected the cells completely.(ABSTRACT TRUNCATED AT 250 WORDS)