The role of antioxidants in the neurotoxicity of the antimalarial endoperoxides artemether and dihydroartemisinin was studied in vitro by quantitative image analysis of neurite outgrowth in the neuroblastoma cell line NB2a. Intracellular glutathione concentrations were measured by high performance liquid chromatography with fluorescence detection. Both dihydroartemisinin (1 microM) and a combination of artemether (0.3 microM) plus haemin (2 microM) significantly inhibited neurite outgrowth from differentiating NB2a cells to 11.5 +/- 11.0% (SD) and 19.6 +/- 15.2% of controls, respectively. The inhibition by artemether/haemin was prevented by the antioxidants superoxide dismutase (109.7 +/- 47.8% of control), catalase (107.0 +/- 29.3%) glutathione (123.8 +/- 12.4%), L-cysteine (88.0 +/- 6.3%), N-acetyl-L-cysteine (107.8 +/- 14.9%), and ascorbic acid (104.3 +/- 12.7%). Dihydroartemisinin-induced neurotoxicity was completely or partially prevented by L-cysteine (99.5 +/- 17.7% of control), glutathione (57.9 +/- 23.4% of control), and N-acetyl-L-cysteine (57.3 +/- 9.5%), but was not prevented by superoxide dismutase, catalase, or ascorbic acid. Buthionine sulphoximine, an inhibitor of gamma-glutamylcysteine synthetase, significantly increased the neurotoxic effect of non-toxic concentrations of artemether/haemin (0.1 microM/2 microM) and dihydroartemisinin (0.2 microM), suggesting that endogenous glutathione participates in the prevention of the neurotoxicity of artemether/haemin and dihydroartemisinin. Artemether/haemin completely depleted intracellular glutathione levels, whereas dihydroartemisinin had no effect. We conclude that although glutathione status is an important determinant in the neurotoxicity of endoperoxides, depletion of glutathione is not a prerequisite for their toxicity. This is consistent with their mechanisms of toxicity being free radical-mediated damage to redox-sensitive proteins essential for neurite outgrowth, or alteration of a redox-sensitive signalling system which regulates neurite outgrowth.