The exact pathogenesis of neuronal death following bleeding in brain parenchyma is still unknown. Hemoglobin (Hb) toxicity has been postulated to be one of the underlying mechanisms. The purpose of this study was to examine the possible contribution to neurotoxicity of each of the Hb compounds and to characterize the death pathway. Pheochromocytoma (PC12) and neuroblastoma (SH- SY5Y) cell lines were exposed to Hb, globin, hemin, protoporphyrin IX and iron for 1.5- 24 h. We found that Hb and hemin are highly toxic (LD(50) of 8 and 20 &mgr; mol/l, respectively) as compared to globin that was not toxic. In addition, protoporphyrin IX and iron, compounds of hemin, were less toxic than hemin itself (LD(50) of 962 and 2070 &mgr; mol/l respectively). We also demonstrated that non-specific protein digestion with proteinase-K, markedly increased Hb toxicity. Hemin-treated cells caused a typical apoptotic cell death pattern as indicated by DNA fragmentation, caspase activation and reduction in the mitochondrial membrane potential. Treatment with the antioxidant N-acetyl-L-cysteine or iron chelator, deferoxamine, diminished hemin-induced cell death, indicating a role of oxidative stress in this deleterious process. Thus, therapeutic strategies, based on antioxidant, iron chelator and anti-apoptotic agents may be effective in counteracting Hb neurotoxicity.