Changes in the oxidant/antioxidant environment of aging Leydig cells have been shown to be correlated with the reduced ability of these cells to produce testosterone. With this in mind, we hypothesized that the experimental depletion of glutathione (GSH), an abundant Leydig cell intracellular antioxidant, might result in reduced testosterone production. Incubation of Leydig cells isolated from the testes of adult Brown Norway rats with buthionine sulfoximine (BSO) reduced GSH content by more than 70%, and testosterone production by about 40%. The antioxidants vitamin E, N-tert-butyl--phenylnitrone and Trolox countered BSO's effect on steroidogenesis but not on GSH depletion. Together, BSO and glutathione ethyl ester maintained intracellular GSH and also testosterone production, while 1,2-dithiole-3-thione, which increases intracellular GSH, increased testosterone production. In vivo studies also were conducted. Young (4 month-old) and old (24 month-old) rats were injected with BSO twice a day for 7 days, after which Leydig cells were isolated and analyzed in vitro. BSO treatment reduced Leydig cell GSH content by 70%, and the ability of the Leydig cells to produce testosterone by more than 50%. As with aging, decreases were seen in LH-stimulated cAMP production, steroidogenic acute regulatory (StAR) protein, cholesterol side-chain cleavage, 3-hydroxysteroid dehydrogenase and 17-hydroxylase/17,20-lyase. The results of these studies, taken together, are consistent with the hypothesis that alteration in the oxidant/antioxidant environment may play a significant, causative role in the age-related reduced ability of Leydig cells to produce testosterone.