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 functionin 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<0.01) less="" g-6-pdh,="" total="" sod,="" glutathione="" reductase="" and="" total="" glutathione,="" but="" significantly=""><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="" gluthathione="" (gsh)="" which="" couples="" with="" glutathione="" peroxidase="" to="" breakdown="" hydrogen="" peroxide="">₂O₂) 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="">₂O₂ 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.