We previously showed, using microdialysis, that autoxidation of exogenous L-dihydroxyphenylalanine (L-DOPA) occurs in vivo in the extracellular compartment of the freely moving rat, with a consequent formation of L-DOPA semiquinone (L-DOPA-SQ). In the present study, intrastriatal infusion of L-DOPA (1.0 microm for 200 min) increased dialysate L-DOPA concentrations (maximum increases up to 116-fold baseline values); moreover, L-DOPA-SQ was detected in dialysates. Individual dialysate concentrations of L-DOPA were negatively correlated with those of L-DOPA-SQ. Co-infusion of N-acetylcysteine (100 microm) or melatonin (50 microm) increased L-DOPA (up to 151- and 246-fold, respectively) and decreased L-DOPA-SQ (by about 53% and 87%, respectively) dialysate concentrations. Systemic L-DOPA [25 mg/kg intraperitoneally (i.p.) twice in a 12-h interval] significantly increased striatal baseline dialysate concentrations of L-DOPA and decreased dopamine (DA) and ascorbic acid (AsAc) concentrations, when compared with controls. Following systemic L-DOPA, L-DOPA-SQ was detected in dialysates. Endogenous melatonin was depleted in rats maintained on a 24-h light cycle for 1 wk. In melatonin-depleted rats, systemic L-DOPA induced a smaller increase in dialysate L-DOPA, a greater increase in L-DOPA-SQ formation, and a greater reduction in DA and AsAc dialysate concentrations. Co-administration of melatonin (5.0 mg/kg, i.p., twice in a 12-h interval) with L-DOPA, in control as well as in light-exposed rats, significantly increased dialysate L-DOPA concentrations, greatly inhibited L-DOPA-SQ formation, and restored up to the control values dialysate DA and AsAc concentrations. These findings demonstrate that endogenous melatonin protects exogenous L-DOPA from autoxidation in the extracellular compartment of the striatum of freely moving rats; moreover, systemic co-administration of melatonin with L-DOPA markedly increases striatal L-DOPA bioavailability in control as well as in melatonin-depleted rats. These results may be of relevance to the long-term L-DOPA therapy of Parkinson's disease.