We have induced intracellular dopamine oxidation to aminochrome in RCSN-3 cells derived from rat substantia nigra by inhibiting VMAT-2 with reserpine to increase free cytosolic dopamine concentration, to study aminochrome-dependent neurotoxicity in the absence of exogenous oxidizing agents such as metals, which may potentiate an aminochrome cytotoxic effect. The expression of VMAT-2 in RCSN-3 cells was determined by reverse transcriptase-polymerase chain reaction and immunocytochemistry. We observed double membrane bodies containing melanin when RCSN-3 cells were incubated with 100 microM dopamine by using transmission electron microscopy. No significant difference in the cell death was observed when the cells were treated 100 microM dopamine and 25 microM reserpine in the absence or presence of 100 microM dicoumarol, an inhibitor of DT-diaphorase. The lack of effect was due to the inhibitory action of 25 microM reserpine on DT-diaphorase (Ki = 24 microM). However, a significant increase in the cell death was observed when DT-diaphorase was inhibited when the cells were incubated with 1 microM reserpine and 100 microM dopamine for 12 h since at this concentration reserpine inhibits VMAT-2 but not DT-diaphorase. Under this condition, we observed (i) the formation of blebbing; (ii) chromatin condensation accompanied by the formation of massive patches in contact with the nuclear membrane; (iii) the smoothness of the cell's surface, that is, lack of surface microprojections; and (iv) mitochondrial damage characterized by disruption of cristae architecture, which remains closely packed; disorganization of the mitochondrial matrix due to separation of the outer membrane from the internal membrane and considerable enlargement of the intermembrane space; and disruption of the external mitochondrial membrane determined by transmission electron microscopy. These results support the proposed neuroprotective role of DT-diaphorase against aminochrome neurotoxicity, and it suggests that RCSN-3 cells incubated with reserpine and dopamine are an excellent and more physiological cellular experimental model to study the role of dopamine oxidation in neurotoxic effects of dopamine.