MPTP is a toxin presumed to damage dopamine-secreting neurons by an oxygen free radical-mediated mechanism. Two steps in MPTP metabolism are the primary candidates for oxygen free radical generation: (a) MPTP oxidation to MPP(+) by a monoamine oxidase and (b) NADH dehydrogenase inhibition by MPP(+). In order to test the idea that MPTP toxicity is mediated by oxygen free radicals, we assessed lipid peroxidation and the effects of antioxidants in dopaminergic PC12 cells treated with MPTP or MPP(+). For comparison purposes, we also examined the effects of the pro-oxidant tert-butyl-hydroperoxide (TBHP) and of the dopaminergic toxin 6-hydroxydopamine (6-OHDA) in PC12 cells. MPTP and MPP(+), unlike TBHP, failed to induce lipid peroxidation in PC12 cells after a 4-h exposure. All toxins tested (MPTP, MPP(+), TBHP and 6-OHDA) caused a dose-dependent decrease in [(3)H]dopamine ((3)H-DA) uptake in PC12 cultures. The hydroperoxide scavengers glutathione and N-acetyl-cysteine and the superoxide and peroxide scavenger EUK-134 protected PC12 cells from TBHP- and 6-OHDA-induced decrease in (3)H-DA uptake. However, no protection by these antioxidants at various concentrations and time regimens was observed against MPTP- or MPP(+)-induced decreases in (3)H-DA uptake in PC12 cells. In addition, incubation of PC12 cells with the energy-rich substrate, NADH, attenuated MPP(+)-induced decrease in (3)H-DA uptake. These results suggest that MPTP-induced toxicity in dopaminergic PC12 cell cultures, does not involve oxygen free radical production, but rather may be caused by impairment in energy metabolism.