Despite the multiple roles played by antioxidants in rhizobia–legume symbioses, little is known about glutathione peroxidases (GPXs) in legumes. Here the characterization of six GPX genes of Lotus japonicus is reported. Expression of GPX genes was analysed by quantitative reverse transcription– polymerase chain reaction in L. japonicus and Lotus corniculatus plants exposed to various treatments known to generate reactive oxygen and/or nitrogen species. LjGPX1 and LjGPX3 were the most abundantly expressed genes in leaves, roots and nodules. Compared with roots, LjGPX1 and LjGPX6 were highly expressed in leaves and LjGPX3 and LjGPX6 in nodules. In roots, salinity decreased GPX4 expression, aluminium decreased expression of the six genes, and cadmium caused up regulation of GPX3, GPX4 and GPX5 after 1 h and down-regulation of GPX1, GPX2, GPX4 and GPX6 after 3–24 h. Exposure of roots to sodium nitroprusside (a nitric oxide donor) for 1 h increased the mRNA levels of GPX4 and GPX6 by 3.3- and 30-fold, respectively. Thereafter, the GPX6 mRNA level remained consistently higher than that of the control. Immunogold labelling revealed the presence of GPX proteins in root and nodule amyloplasts and in leaf chloroplasts of L. japonicus and other legumes. Labelling was associated with starch grains. These results underscore the differential regulation of GPX expression in response to cadmium, aluminium and nitric oxide, and strongly support a role for GPX6 and possibly other GPX genes in stress and/or metabolic signalling.