Glutathione (GSH) is one of the most important antioxidants involved in detoxification of reactive oxygen and nitrogen species. We investigated the changes in intracellular GSH in primary glial cultures stimulated to produce inducible nitric oxide synthase and subsequently nitric oxide by bacterial lipopolysaccharide and gamma interferon treatment. Intracellular GSH content was measured by both the monochlorobimane fluorescence microscopy method and the GSH reductase recycling assay (Tietze. Anal Biochem 27:502-522, 1969.). Our results show that irrespective of the assay used the GSH content in stimulated cultures decreased to almost half that of control cultures. This decrease in GSH content was accompanied by an increase in S-nitrosoglutathione in the stimulated cultures. Analysis of the GSH related fluorescence images showed that the fluorescence intensity was lowered exclusively in microglial cells whereas that of astrocytes remained almost unchanged. The present study in conjunction with our previous investigation (Chatterjee et al. Glia 27:152-161, 1999) can be interpreted to imply that the higher GSH levels in untreated microglia are a mechanism to withstand nitric oxide synthase induced oxidative and nitrosative stress and therefore the GSH levels in microglia drop to astrocyte levels after induction.