SummaryThe different mechanisms for glutathione‐dependent inactivation of a number of oxidizing compounds and other xenobiotics were studied using isolated round spermatids from rats. For the estimation of cellular GSH a flow cytometric assay was used. The cells were exposed to the oxidizing compounds cumene hydroperoxide and diamide, to study the activity of the GSH redox cycle. Incubation of the isolated cells with these compounds showed that the cells had a limited capacity to withstand the oxidative stress associated with their inactivation. The GSH level of the spermatids was maintained during 18 h of incubation in the presence of low concentrations of cumene hydroperoxide and diamide, whereas spermatids exposed to higher concentrations showed a loss of both GSH and ATP. No partial loss of GSH from individual cells was observed. Diethyl maleate and 1,2‐epoxy‐p‐(nitrophenoxy)propane (ENPP) were used to study the effect of glutathione S‐transferase‐catalysed GSH conjugation on the GSH content of spermatids. Exposure of the cells to low concentrations of diethyl maleate and ENPP resulted in a decrease in GSH content. The flow cytometric analysis showed that this was a partial loss of GSH from all cells, rather than GSH depletion in a part of the cell population. This diminution of the cellular GSH pool, however, did not affect the ATP content and viability of the cells. The present results indicate that spermatids can utilize GSH‐dependent defence mechanisms against a number of mo