Multiple mechanisms have been proposed to contribute to amyotrophic lateral sclerosis (ALS) pathogenesis, including oxidative stress. Early evidence of a role for oxidative damage was based on the finding, in patients and murine models, of high levels of markers, such as free nitrotyrosine (NT). However, no comprehensive study on the protein targets of nitration in ALS has been reported. We found an increased level of NT immunoreactivity in spinal cord protein extracts of a transgenic mouse model of familial ALS (FALS) at a presymptomatic stage of the disease compared with age-matched controls. NT immunoreactivity is increased in the soluble fraction of spinal cord homogenates and is found as a punctate staining in motor neuron perikarya of presymptomatic FALS mice. Using a proteome-based strategy, we identified proteins nitrated in vivo, under physiological or pathological conditions, and compared their level of specific nitration. alpha- and gamma-enolase, ATP synthase beta chain, and heat shock cognate 71-kDa protein and actin were overnitrated in presymptomatic FALS mice. We identified by matrix-assisted laser desorption/ionization mass spectrometry 16 sites of nitration in proteins oxidized in vivo. In particular, alpha-enolase nitration at Tyr(43), target also of phosphorylation, brings additional evidence on the possible interference of nitration with phosphorylation. In conclusion, we propose that protein nitration may have a role in ALS pathogenesis, acting directly by inhibiting the function of specific proteins and indirectly interfering with protein degradation pathways and phosphorylation cascades.