The vulnerability of spinal cord neurons to hemoglobin was quantitatively assessed in primary cultures derived from fetal mice. Exposure to hemoglobin for 28 h in a serum-free medium resulted in concentration-dependent neuronal death, with an EC50 of 0.9 microM; glia were not injured. Neuronal death was decreased by the ferric iron chelator deferoxamine, the alpha-tocopherol analogue Trolox C, ascorbate, and exogenous catalase, but was potentiated by superoxide dismutase. Neuronal death was also increased by depletion of cellular glutathione with the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoxamine; inhibition of endogenous catalase with 3-amino-1,2,4-triazole had no significant effect. These results suggest that hemoglobin is toxic to spinal neurons via an iron-dependent, oxidative mechanism involving a hydrogen peroxide intermediate, and support the hypothesis that hemoglobin release may contribute to neuronal loss after spinal cord trauma.