Copper (Cu) in excess can disturb the cell redox status maintained by reactive oxygen- (ROS) and nitrogen species. With the help of the nitric oxide (NO)-deficient nia1nia2noa1-2 mutant, the role of NO in copper stress tolerance and its relationship with ROS was examined. Under control conditions and also during Cu exposure, the NO level in the cotyledon and root tip of the mutant was significantly lower compared to the wild-type (WT) suggesting the contribution of the nitrate reductase- and nitric oxide associated 1-dependent pathways to NO synthesis. The cell viability decrease was more pronounced in the triple mutant and the originally low growth rate was maintained under Cu stress. The endogenous NO level of the mutant was increased by NO donor addition and its cell viability significantly improved suggesting that the Cu sensitivity of the nia1nia2noa1-2 mutant is directly associated with its low NO content. As the effect of Cu increased ROS formation occurred in WT roots, while the originally high ROS levels of the triple mutant slightly decreased, still remaining significantly higher than those in the WT. In the cotyledons of the triple mutant 5 µM Cu induced ROS production but NO formation failed, while in the WT cotyledons NO but no ROS accumulation was observed. The promoting effect of NO deficiency on ROS production assumes an antagonism between these molecules during Cu stress. Based on the results, it can be concluded that NO contributes to copper tolerance and its deficiency favours for ROS production.