Here, we report that an E-box element located within the human topoisomerase III alpha (hTOP3 alpha) gene promoter acts as a cell type-specific enhancer. The upstream stimulatory factor (USF) was shown to specifically recognize the mutationally sensitive E-box element. When assayed by transient transfection with hTOP3 alpha promoter-dependent reporter genes, USF is transcriptionally active in HeLa cells but lacks transcriptional activity in Saos-2 cells. The hTOP3 alpha mRNA level in Saos-2 cells was reduced to about 30% of the level observed for HeLa cells, suggesting that the inactivity of USF in hTOP3 alpha promoter activity may be the cause of the marked reduction of hTOP3 alpha mRNIA levels in Saos-2 cells. Using transient transfection assays in HeLa cells, we demonstrated that ectopically expressed USF2, but not USF1, was capable of activating hTOP3 alpha transcription through the E-box element. However, USF2 did not stimulate hTOP3 alpha promoter activity in Saos-2 cells. This cell type-specific regulation of promoter activity by USF2 may provide a mechanism for the differential expression of hTOP3 alpha in various tissues and during developmental stages.