Leghemoglobins together with high rates of respiration are believed to be major sources of reactive oxygen species (ROS) in root nodules of leguminous plants. High capacities of antioxidative systems apparently protect this organ from oxidative damage. Using leghemoglobin-RNA interference (LbRNAi) lines of Lotus japonicus, we found that loss of leghemoglobin results in significantly lower H2O2 levels in nodules. Transcript levels and catalytic activities of ascorbate-glutathione cycle enzymes involved in H2O2 detoxification as well as concentrations of reduced ascorbate were also altered in LbRNAi nodules. Thus, symbiotic leghemoglobins contribute significantly to ROS generation in functional nodules.