Drought is considered one of the abiotic stresses with significant implications on plant productivity. Previously, we have shown that water deficit produces a differential nitro-oxidative stress in roots and leaves of Lotus japonicus L. plants. Using this model legume, we studied the nitro-oxidative stress in drought-stressed roots by complementary biochemical, cellular and proteomic approaches. Cellular analyses of root cross-sections by confocal laser scanning microscopy (CLSM) using specific fluorescent probes for superoxide radical (O-2(center dot-)), nitric oxide (NO), peroxynitrite (ONOO-) and S-nitrosothiols (SNOs) showed that drought stress causes a differential cellular localization of these reactive species. Mainly,O-2(center dot-) and ONOO- had a wide distribution in almost all root cell types (xylem, parenchyma, and peridermis), whereas NO and SNOs accumulated in cortical cells (peridermis). Liquid chromatography-electrospray/mass spectrometry (LC-ES/MS) analyses showed that the content of ascorbate, S-nitrosoglutaathione (GSNO), and reduced glutathione (GSH) in drought-stressed roots was drastically diminished. Nitroproteome analysis by two-dimensional gel electrophoresis and mass spectrometry allowed to identify 13 tyrosine-nitrated proteins such as methionine synthase, Hsp70, adenosyl-homocysteinase, peroxidase, alcohol dehydrogenases, glutamine synthetase, fructokinase, 1,3-beta-glucanase, chitinases, endochitinase, among others which are directly (24%) or indirectly (74%) related to plant defense. Taken together, these results indicate that drought-stressed roots have an active metabolism of reactive oxygen and nitrogen species (ROS and RNS) characterized by an increase of protein nitration and accumulation of NO and SNOs in cortical cells. The possibility of autophagy taking place in the stressed roots is also discussed.