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The mechanism of copper (Cu) neurotoxicity was studied in the RCSN-3 neuronal dopaminergic cell line, derived from substantia nigra of an adult rat. The formation of a Cu–dopamine complex was accompanied by oxidation of dopamine to aminochrome. We found that the Cu–dopamine complex mediates the uptake of [sup 64]CuSO[sub 4] into the Raúl Caviedes substantia nigra-clone 3 (RCSN3) cells, and it is inhibited by the addition of excess dopamine (2 m m) (63%, p < 0.001) and nomifensine (2 µm) (77%, p < 0.001). Copper sulfate (1 m m) alone was not toxic to RCSN-3 cells, but was when combined with dopamine or with dicoumarol (95% toxicity; p < 0.001) which inhibits DPNH and TPNH (DT)-diaphorase. Electron spin resonance (ESR) spectrum of the 5,5-dimethylpyrroline-N-oxide (DMPO) spin trap adducts showed the presence of a C-centered radical when incubating cells with dopamine, CuSO[sub 4] and dicoumarol. A decrease in the expression of CuZn-superoxide dismutase and glutathione peroxidase mRNA was observed when RCSN-3 cells were treated with CuSO[sub 4], dopamine, or CuSO[sub 4] and dopamine. However, the mRNA expression of glutathione peroxidase remained at control levels when the cells were treated with CuSO[sub 4], dopamine and dicoumarol. The regulation of catalase was different since all the treatments with CuSO[sub 4] increased the expression of catalase mRNA. Our results suggest that copper neurotoxicity is dependent on: (i) the formation of Cu–dopamine complexes with concomitant dopamine oxidation to aminochrome; (ii) dopamine-dependent Cu uptake; and (iii) one-electron reduction of aminochrome. [ABSTRACT FROM AUTHOR]