The posttranscriptional regulation of GLUT1 glucose transporter gene expression may be mediated by specific interactions between cytosolic trans-acting factors and regulatory cis-elements within the 3'-untranslated regions (UTRs) of the GLUT1 mRNA. Recent studies demonstrate that experimental and human brain tumors express an 80-kDa protein that reacts with a specific sequence around nucleotide 2,200 within the GLUT1 mRNA 3'-UTR. The 80-kDa protein is selectively expressed in hemangioblastoma, a tumor characterized by overexpression of GLUT1. The enhancer role of this GLUT1 3'-UTR cis-element was confirmed in the present studies using the luciferase expression vector pGL2 and site-directed deletion. Transfection of C6 glioma cells with pGL2 (containing nucleotides 2,100-2,300 of the bovine GLUT1 3'-UTR inserted at the Pfl MI site within the luciferase 3'-UTR) results in a fivefold increase in luciferase gene expression. Deletion of nucleotides 2,181-2,190 of the bovine GLUT1 3'-UTR, i.e., the putative binding site of the 80-kDa protein, completely eliminated the enhancement of luciferase activity in the transfected cells. Luciferase mRNA containing the putative cis-element inserted in the 3'-UTR was transcribed, and after UV crosslinking, this mRNA complexed with the 80-kDa protein in cytosol of either C6 cells or hemangioblastoma. In contrast, this complex was undetected with either luciferase control mRNA or 10 nucleotide-deleted RNA. The present study provides evidence that nucleotides 2,181-2,190 of the bovine GLUT1 mRNA 3'-UTR forms a complex with brain tumor cytosolic proteins that serves to increase GLUT1 gene expression at the posttranscriptional level.