Human upstream binding factor (hUBF) is a sequence-specific DNA-binding protein that is essential for the activation of human 18s and 28s rRNA gene transcription. We have isolated and localized the gene (UBTF) encoding hUBF to the D17S183 locus on chromosome 17q21 by analyzing a cosmid from the region and carrying out Southern analysis on a previously constructed chromosome 17 somatic cell hybrid mapping panel using a probe from the hUBF cDNA. Confirmation of its location at this region was obtained from the results of pulsed-field gel electrophoresis analysis of genomic DNA using the hUBF cDNA and other probes from the region. These data also enabled the construction of a long-range restriction map of the region.