Peroxynitrite formation has been demonstrated in several neurodegenerative disorders; thus far, protein nitration and consequent alterations in protein function are implicated as mechanistic events. Free 3-nitrotyrosine (free-3NT) is also elevated in these settings; a neurotoxic role for this modified amino acid has not been investigated. We tested the hypothesis that free-3NT is neurotoxic in vivo, using a mouse model of striatal degeneration. The neurodegenerative effects of the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) (unilateral intrastriatal injection, 64 nmol) were compared with free-3NT (32 nmol) or free-tyrosine (free-TYR) (32 nmol). 6-OHDA-treated mice exhibited significant ipsilateral turning behavior after d-amphetamine challenge, indicative of unilateral striatal injury (ipsilateral-contralateral turning differential, 21.1 +/- 6.8). Significant turning behavior was also observed in free-3NT-treated mice but not in free-tyrosine-treated mice (free-3NT, 16.0 +/- 3.9; free-TYR, 1 +/- 2.7; p < 0.01). Immunohistochemistry was used to evaluate striatal tyrosine hydroxylase (TH) content. 6-OHDA or free-3NT treatment caused severe reductions in TH immunoreactivity in injected striata compared with the contralateral hemisphere (injected/contralateral immunoreactivity ratio: 6-OHDA, 0.23 +/- 0.07; free-3NT, 0.49 +/- 0.02). Free-tyrosine treatment had no effect (1.03 +/- 0.09). Turning behavior was correlated with striatal TH ratio (p < 0.01). Furthermore, we observed a striking unilateral reduction in TH-positive cell body counts in the substantia nigra pars compacta of 6-OHDA- and free-3NT-treated mice (injected/contralateral cell count ratio: 6-OHDA, 0.40 +/- 0.04; free-3NT, 0.59 +/- 0.02). Free-tyrosine treatment had no effect (1.05 +/- 0.04). No evidence for increased striatal protein incorporation of 3NT was observed in any treatment group. These data represent the first evidence that free-3NT can elicit neurodegenerative effects in vivo; free-3NT may have a causal role in neurodegenerative conditions.