Divalent metal transporter 1 (DMT1), expressed in many different tissues, is responsible for the transport of a broad range of divalent metal ions. DMT1 exists in at least, four distinct isoforms which differ in both the C-terminus (termed here (-)IRE and (+)IRE) and the N-terminus (transcription proceeds from two different promoters). In the rat, two of the forms possess an additional 31 amino acids in the N-terminus (termed exon 1A) whereas the shorter forms lack this sequence (termed exon 2). Studies were performed to compare differences in expression and localization of these isoforms in low density and confluent cultures of rat astrocytes obtained from traumatized striatum and in rat C6 astrocytoma and human U87 glioblastoma. Results of these experiments reveal the presence of both the (+/-)IRE forms of DMT1 in all cultured cells examined. Western blots using affinity purified antibodies, which differentially recognize the two C-terminal species of DMT1, indicate a strong upregulation of the (+)IRE form in low density astrocyte cultures when compared to confluent cultures. Previously we reported that the (-)IRE form was present in both the nucleus and cytoplasm in neurons and neuronal like cells whereas the (+)IRE form was exclusively cytoplasmic. Similar results were found with the (-)IRE species in astrocytes and astrocytomas, i.e. nuclear and cytoplasmic distribution. This form of DMT1 also colocalizes with the early endosomal marker, EEA, suggesting that (-)IRE species may function in the transport of divalent metals. In contrast to our previous findings, however, the (+)IRE form was found predominantly localized in nucleus in both the primary and neoplastic glial cells. Interestingly, neither form of DMT1 colocalizes with the transferrin receptor. These data suggest that selective compartmentalization of specific isoforms of DMT1 imparts distinct and specialized functions that meet the changing needs of essential divalent transition metals as cofactors within cells.