Exposure to hyperoxia causes alveolar macrophage (AM) injury. The present study investigates the roles of intracellular antioxidant enzymes and of glutathione in the protection of AMs against hyperoxia in a biphasic cell culture system in aerobiosis. The effect of normoxia or hyperoxia on the integrity of AMs was related to indices of cell injury (ATP cell content and lactate dehydrogenase release into culture medium) and cell mass (protein content of AMs). Antioxidant activities were measured in guinea-pig AMs exposed to 95% O2 or to normoxia (control cells) for 3 days. A 3-day AM culture in normoxia showed a significant decrease in protein and catalase, whereas ATP cell content, superoxide dismutase (SOD) (both Cu,Zn-SOD and Mn-SOD) and glutathione peroxidase (GPx) activities significantly increased. The content of reduced glutathione (GSH) did not change. Using the ATP content in AMs expressed as a cell injury index (CII), AM injury increased with increasing 0, exposure time (1 day: 13 2 4.4%; 2 days: 34 * 3.8%; 3 days: 40 rt-4.1%; 4 days: 55 2 7.3%; 6 days: 87.5 2 5.4%). Exposure to 95% O2 for 3 days was associated with a significant decrease in ATP cell content, protein, catalase and GSH to the total glutathione ratio, whereas SOD, GSH and total glutathione did not change significantly. The GPx activities increased significantly. There was no significant correlation between the AM CII and SOD or GPx content. In contrast, a significant correlation was observed between hyperoxia-induced AM CII and catalase content (r = 0.71) and glutathione content (r = 0.71). Incubation of AMs with buthionine sulphoximine, a specific inhibitor of glutathione synthesis, significantly decreased the GSH content, whereas the ATP content and antioxidant enzyme activities in the AMs did not change. Reduction of intracellular GSH increased hyperoxia (50% 0,)-induced AM injury from 2.2 rt-6% to 53 rt-10.2%. Our results support the concept that regulation of cell GSH is an important mechanism of protection against hyperoxic injury.