Enhanced expression of human telomerase reverse transcriptase (hTERT) occurs frequently during cellular immortalization. The current study was undertaken to determine the mechanism regulating the hTERT promoter activity during cellular immortalization of human oral keratinocytes. Normal human oral keratinocytes (NHOKs) were immortalized with Bmi-1 and the E6 oncoprotein of human papillomavirus type 16 to establish the telomerase-positive HOK-Bmi-1/E6 cell line. Using DNA-protein-binding assay, we found that heat shock protein 90 (hsp90) physically interacts with the hTERT promoter in vitro. The hsp90 interaction with the promoter was detected more strongly in the telomerase-positive HOK-Bmi-1/E6 cells compared with that in senescing NHOK. Chromatin immunoprecipitation confirmed the in vivo interaction between hsp90 and the hTERT promoter in SCC4 cells, a telomerase-positive oral cancer cell line, but not in the NHOK. To determine the physiological significance of this interaction, SCC4 cells were exposed to geldanamycin (GA), a competitive inhibitor of hsp90. GA exposure led to decrease in telomerase activity, hTERT promoter activity and hTERT messenger RNA expression in SCC4 cells, even in the absence of de novo protein synthesis. Also, it abolished the in vivo interaction of the hTERT promoter region with hsp90 but not with Sp1 or c-Myc. These results indicate that physical interaction between hsp90 and the hTERT promoter occurs in telomerase-positive cells but not in normal human cells and is necessary for the enhanced hTERT expression and telomerase activity in cancer cells.