Tumor susceptibility gene 101 (TSG101), a mammalian homologue of yeast vps23, is involved in protein sorting, vesicular trafficking and maintenance of genomic integrity. Upregulation of the TSG101 gene was found in human thyroid papillary and breast tumors. Here, we define the proximal promoter of human TSG101 at -1 to -436 by reporter assay. Intact Sp1 and MAZ binding sequences within this region are essential, and mutation of both sites eliminates proximal promoter activity implying cooperation between these two cis-elements. Chromatin immunoprecipitation and DNA affinity precipitation assay confirmed in vivo Sp1 binding on the GGGGCGGGTT sequence. MAZ protein was essential for TSG101 promoter activity because its knockdown using siRNA decreased reporter activity. An upstream regulatory element (URE) at the -1280 to -1757 region was identified to confer the orientation-independent enhancement of the promoter activity in transformed COS-1, ARO and WRO cell lines but not in a normal thyroid FRTL cell line. The sequence of this URE region contains putative binding sites for thyroid transcription factor 2 (TTF-2) and thyroid hormone receptor (T3R), which might be relevant to differential regulation of TSG101 promoter activity in transformed and primary cells.