Hemoglobin (Hb) has been demonstrated to be neurotoxic when injected into the cerebral cortex in vivo. However, associated systemic factors such as ischemia and epileptogenesis have limited investigations of Hb toxicity in the intact central nervous system (CNS). In this study, the neurotoxicity of human Hb was assessed in mixed neuronal and glial neocortical cell cultures derived from fetal mice. Exposure of cultures to Hb for 24–28 h produced widespread and concentration-dependent neuronal death (EC₅₀ 1–2.5 μM), without injuring glia. Brief exposures (1–2 h) were not toxic. Neuronal death was completely blocked by the 21-aminosteroid U74500A, the antioxidant Trolox, and the ferric iron chelator deferoxamine. The results of these experiments suggest that, in this system, Hb is a potent neurotoxin, and that Hb neurotoxicity may contribute to secondary injury processes after trauma and intracranial hemorrhage.