Activation of murine microglial and macrophage cell lines with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) resulted in the induction of the inducible form of nitric oxide synthase (NOS) and the release of micromolar amounts of NO into the surrounding medium. The synthesis of NO was associated with increased cellular membrane damage as assessed by trypan blue dye exclusion and the leakage of lactate dehydrogenase into the cell culture medium. However, the synthesis and release of cytokines was largely unaffected. NO-mediated cell damage was also accompanied by a marked decrease in the intracellular levels of reduced glutathione and ATP. In addition, significant inhibition of mitochondrial respiratory chain enzyme activities was seen following cellular activation. However, citrate synthase activity (a mitochondrial matrix enzyme) was not detectable in the extracellular supernatants, suggesting preservation of the integrity of the mitochondrial inner membrane following activation. These effects were largely prevented by the addition of the NOS inhibitor, N-guanidino monomethyl l-arginine during the activation period. Our observations demonstrate that induction of NOS activity in microglia results in damage to the plasma membrane leading to a loss of glutathione, complex-specific inhibition of the mitochondrial electron transport chain and depletion of cellular ATP. Our data suggest that pharmacological modulation of NOS activity in activated microglia in vivo may prevent cellular damage to bystander cells such as neurons, astrocytes and oligodendrocytes, as well as to microglia themselves.