The 90-kDa nucleolus organizer region autoantigen (NOR-90) was previously shown to be identical to human upstream binding factor (hUBF), which has two molecular mass forms of 89 and 93 kDa, respectively. hUBF/NOR-90 is a member of the HMG-box DNA-binding protein family and is known to bind to enhancer regions upstream of the ribosomal RNA genes, which are clustered at NORs. The smaller version of UBF lacks an internal 111-bp region corresponding to 37 amino acids in the second HMG-box of the larger form. We isolated human genomic clones from a phage library and localized one of them by fluorescence in situ hybridization to chromosome 17q21.3. DNA sequence analysis showed that the 111-bp region represented a single exon, consistent with the previous notion that the two isoforms were products of alternative pre-mRNA splicing of a single gene in human. Radiation hybrid mapping placed this STS with very high probability (LOD > 19) to chromosome 17, approximately 3.77 cR distal to MIT framework marker UTR-9641. The order of the markers (a partial list) from this region was UTR-9641, SGC30031, WI-17308, D17S930, NOR53/33, WI-16100, D17S920, WI-16913, and WI-6808.