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Utilization of highly enriched preparations of steroidogenic Leydig cells have proven invaluable for studying the direct effects of various hormones and agents on Leydig cell function in vitro. However, recent work indicates that isolated Leydig cells are often subjected to oxygen O2 toxicity when cultured at ambient 19% oxygen concentrations. Because intracellular antioxidants play an important role in protecting cells against oxygen toxicity, we have investigated the intracellular antioxidant defense system of isolated Leydig cells. The cellular levels of several antioxidants including catalase, glucose-6-phosphate dehydrogenase G-6-PDH, superoxide dismutase SOD of the Cu/Zn & Mn variety, glutathione peroxidase, glutathione reductase and total glutathione were quantitated using enriched populations of Leydig cells isolated from adult male guinea pig testes. Compared to whole testicular homogenates, Leydig cells contained significantly P &lt; 0.01 less G-6-PDH, total SOD, glutathione reductase and total glutathione, but significantly P &lt; 0.001 more glutathione peroxidase. Compared to hepatic values previously reported in the guinea pig, Leydig cells contain nearly 400 times less catalase, about 14 times less glutathione peroxidase and almost 11 times less glutathione reductase. Since G-6-PDH and glutathione reductase are both necessary to regenerate reduced glutathione GSH which couples with glutathione peroxidase to breakdown hydrogen peroxide H2O2 under normal conditions, it is plausible that the oxygen toxicity observed in isolated Leydig cells is due to the intracellular accumulation of H2O2. Using the dichlorofluorescin diacetate DCF-DA assay, we found that Leydig cells incubated in the presence of 19% O2 produced significantly P<0.001 higher levels of H2O2 with time in culture compared to Leydig cells maintained at 3% O2. These results support the hypothesis that the increased susceptibility of isolated Leydig cells to oxygen toxicity may be due, in part, to decreased amounts of certain antioxidant defenses and an increased production of the reactive oxygen species H2O2.