The protective mechanism of nitric oxide (NO) in regulating tolerance to Cu-induced toxicity in shoots of barley (Hordeum vulgare L.) was studied. The experiment consisted of four treatments based on additions to basal nutrient solutions (BNS): control (CTR), Cu (200 mu M), SNP (500 mu M), and Cu (200 mu M) + SNP (500 mu M) over a period of 10 days. Treatment with Cu significantly reduced seedling growth and photosynthetic efficiency concomitant with an increase in reactive oxygen species contents, lipid peroxidation markers, and antioxidant enzyme activities, indicating that Cu induced oxidative stress. Furthermore, growth inhibition of Cu-treated plants was associated with a reduction in photosynthetic pigments and maximum photosystem II efficiency as well as a strong decrease in levels of glutathione (GSH) and ascorbate (AsA). Addition of a nitric oxide (NO) donor, sodium nitroprusside (SNP), to the growth medium alleviated Cu toxicity by decreasing Cu uptake and enhancing antioxidant capacity, as indicated by increased contents of GSH and AsA. The application of SNP decreased oxidative stress and lipid peroxidation by suppressing lipoxygenase activity and enhancing some antioxidant enzyme activities. The results obtained indicate the potential of exogenously applied SNP in the management of metal toxicity. Hence, NO generating compounds have potential agronomical applications when cultivating in contaminated areas. Our findings indicate that NO can alleviate Cu toxicity by affecting the antioxidant defense system and maintaining the glutathione-ascorbate cycle status, suggesting that SNP treatment protects proteins against oxidation by regulating the cellular redox homeostasis.