We have tested the idea that oxidative metabolism of dopamine may be involved in MPTP toxicity using the RCSN-3 cell line derived from the substantia nigra of an adult rat. Treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (10 microM), MPTP combined with 40 microM dicoumarol (an inhibitor of DT-diaphorase) and dicoumarol alone, did not induce toxicity in RCSN-3 cells after 72 h incubation. The lack of toxicity in MPTP-treated RCSN-3 cells may be explained by the fact that they are unable to metabolize MPTP to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion (MPP+ as determined by HPLC. Incubation for 72 h with 100 microM MPP+ induced a 6.6 +/- 1.4% cell death of RCSN-3 cells compared to 3.5 +/- 0.4 observed in control cells. However, when the cells were treated with 100 microM MPP+ and 40 microM dicoumarol, cell death increased 4-fold compared to that of cells treated solely with MPP+ (27 +/- 2%; P<0.001). Under these conditions, a significant increase in DNA fragmentation (3-fold compared to MPP+ alone; P<0.01) and in calpain activation (P<0.05 compared to control) was evident. The inhibition of DT-diaphorase by dicoumarol supports the idea that oxidative metabolism of dopamine is involved in MPP+ toxicity in RCSN-3 cells.