Astrocytes are central to iron and ascorbate homoeostasis within the brain. Although NTBI (non-transferrin-bound iron) may be a major form of iron imported by astrocytes in vivo, the mechanisms responsible remain unclear. The present study examines NTBI uptake by cultured astrocytes and the involvement of ascorbate and DMT1 (divalent metal transporter 1). We demonstrate that iron accumulation by ascorbate-deficient astrocytes is insensitive to both membrane-impermeant Fe(II) chelators and to the addition of the ferroxidase caeruloplasmin. However, when astrocytes are ascorbate-replete, as occurs in vivo, their rate of iron accumulation is doubled. The acquisition of this additional iron depends on effluxed ascorbate and can be blocked by the DMT1 inhibitor ferristatin/NSC306711. Furthermore, the calcein-accessible component of intracellular labile iron, which appears during iron uptake, appears to consist of only Fe(III) in ascorbate-deficient astrocytes, whereas that of ascorbate-replete astrocytes comprises both valencies. Our data suggest that an Fe(III)-uptake pathway predominates when astrocytes are ascorbate-deficient, but that in ascorbate-replete astrocytes, at least half of the accumulated iron is initially reduced by effluxed ascorbate and then imported by DMT1. These results suggest that ascorbate is intimately involved in iron accumulation by astrocytes, and is thus an important contributor to iron homoeostasis in the mammalian brain.