Hemin is a breakdown product of the blood protein, hemoglobin and is responsible for much of the secondary damage caused following a hemorrhagic stroke. Hemin is toxic to cultured astrocytes and it is thought that this toxicity is due to iron that is liberated when hemin is degraded. However, free iron applied to astrocytes is not toxic and the reason for this discrepancy is unknown. The present study exposed primary astrocyte cultures from neonatal mice to hemin-iron (25 µM hemin) or non-hemin iron (25 µM ferric ammonium citrate; FAC) for 12 or 24 h. Perls' and Turnbull's staining, as well as measures of cell viability and iron accumulation, were used to assess the valency, solubility and distribution of iron within cells. While cells accumulated similar amounts of iron from both sources, hemin was shown to be highly toxic to astrocytes, whereas FAC was not. Iron released by the degradation of hemin was present in both valencies (Fe(2+) and Fe(3+)), was mostly soluble and did not induce ferritin expression in most cells, whereas non-hemin iron (from FAC) was present in astrocytes almost exclusively as insoluble Fe(3+) and it induced widespread ferritin expression. These results show that the cellular mechanisms for processing hemin-iron and non-hemin iron are very different. The data suggest that hemin-iron has a greater potential to damage astrocytes by participating in unregulated redox reactions.