Nitric oxide (.NO) is a reactive nitrogen species known to be involved in cytotoxic processes. Cells respond to cytotoxic injury by stress response induction leading to the development of cellular resistance. This report describes an .NO-induced stress response in Chinese hamster fibroblasts (HA1), which leads to glutathione synthesis-dependent resistance to H2O2-mediated oxidative stress. The development of resistance to H2O2 was completely abolished by the inhibition of glutamate cysteine ligase (GCL) during the first 8 h of recovery after .NO exposure. Altered thiol metabolism was observed immediately after .NO exposure as demonstrated by up to 75% decrease in intracellular thiol pools (glutathione, γ-glutamylcysteine, and cysteine), which then reaccumulated during the .NO-mediated development of resistance. Immunoreactive protein and activity associated with GCL decreased immediately after exposure to .NO and then reaccumulated during the development of resistance to H2O2 challenge. Moreover, compared to N2 controls the activity levels of GCL in .NO-exposed cells increased approximately twofold 24 h after H 2O2 challenge. These results demonstrate that .NO exposure is capable of inducing an adaptive response to H 2O2-mediated oxidative stress in mammalian cells, which involves alterations in thiol metabolism and is dependent upon glutathione synthesis and increased GCL activity. © 2005 Elsevier Inc. All rights reserved.