The present study evaluated the ability of salicylic acid (SA) to attenuate long-term l-DOPA-induced 6-hydroxydopamine (6-OHDA) formation in the striatum of mice, and to protect against the resulting dopaminergic neurotoxicity. The production of 6-OHDA from dopamine in vitro from ferrous-ascorbate-dopamine (FAD) hydroxyl radical ( • OH) generating system or in vivo in the striatum following prolonged administration of l-DOPA in mice were found to be significantly attenuated by SA. Intra-median forebrain bundle infusion of FAD, but not equivalent dose of ferrous ion or dopamine individually, caused significant striatal dopamine depletion, which was blocked by SA administration. The dose- and time-dependent increase in the formation of 6-OHDA following l-DOPA treatment in the mouse striatum was synergistically enhanced to the systemic administration of the parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. SA treatment significantly attenuated the l-DOPA plus the parkinsonian neurotoxin-induced striatal 6-OHDA generation, and protected against striatal dopamine loss. The present study demonstrated a novel mode of dopaminergic neuroprotection by SA and its possible therapeutic implication in the treatment of Parkinson's disease.