The implication of reactive oxygen species for the Ca2+ ionophore ionomycin-induced apoptosis was investigated in cultured cortical neurons from embryonic rats. Ionomycin increased the production of intracellular peroxides as measured by flow cytometric analysis with 6-carboxy-2'7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester). Low doses of ionomycin increased the level of manganese-superoxide dismutase (Mn-SOD). In addition, N-acetyl-L-cysteine prevented apoptotic neuronal death induced by ionomycin in a dose-dependent manner. Buthionine sulfoximine suppressed the effect of N-acetyl-L-cysteine. These results suggest that peroxides and redox-regulation play an important role in the apoptosis of neurons induced by elevation of intracellular Ca2+ concentration. (C) 1996 Academic Press, Inc.