In the present study, we investigated the effect of the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) on hydroxyl free radical and peroxynitrite formation in vivo using D-phenylalanine as a novel mechanistic probe. In vivo microdialysis was carried out in the striatum of freely moving male Wistar rats. The microdialysis probes were perfused with artificial cerebrospinal fluid containing 5 mM D-phenylalanine (flow rate 2 microL/min). After obtaining a stable baseline 6-OHDA was delivered into the striatum via reverse microdialysis for 60 min. HPLC measurements of the effluent were performed using photodiode array detection for determination of phenylalanine derived o-tyrosine and m-tyrosine (as hydroxylation markers) as well as of nitrotyrosine and nitrophenylalanine (as nitration markers). The basal levels of the hydroxylation derived products of phenylalanine were approximately 100-fold higher than those of the nitration derived products. 6-OHDA (0.1, 1, 10 mM) significantly increased o- and m-tyrosine up to nine- and 13-fold, respectively, whereas levels of 3-nitrotyrosine and 4-nitrophenylalanine were significantly increased up to 422- and 358-fold, respectively. The results demonstrate that phenylalanine is a sensitive in vivo marker for 6-OHDA-induced hydroxylation and nitration reactions which are clearly concentration dependent. We conclude that peroxynitrite formation is involved in 6-OHDA-induced neurochemical effects.