Transferrin (Tf) donates iron (Fe) to the brain by means of receptor-mediated endocytosis of Tf at the brain barriers. As Tf transport through the brain barriers is restricted, Fe is probably released into the brain extracellular compartment as non-Tf-bound iron (NTBI). To evaluate NTBI in the brain and cerebrospinal fluid (CSF), different aged rats (P15, P20, P56) were injected intravenously with [59Fe-125I]Tf followed by sampling of CSF and brain tissue. Between 80 and 93% of 59Fe in CSF was absorbed with anti-Tf and 1 and 5% with anti-ferritin antibodies. The fraction of 59Fe from CSF passing through a 30,000 molecular weight (MW) cutoff filter was approximately 5% (P15), 10% (P20), and 15% (P56). Measurements of Fe and Tf concentrations in CSF of P20 rats revealed that the Fe-binding capacity of Tf was exceeded. In the supernatants of brain homogenates, between 94 and 99% of 59Fe was absorbed with anti-Tf and anti-ferritin antibodies. The respective fractions of 59Fe in the supernatants passing through the 30 kD cutoff filter were 4% (P15), 2% (P20), and 6% (P56). In brain homogenates mixed before filtering with desferroxamine (DFO) or nitrilotriacetic acid (NTA) which complex loosely protein-bound Fe and non-protein-bound Fe, these 59Fe fractions were 2-fold higher. The results indicate that NTBI is present extracellularly in CSF and probably in brain interstitium.