The expression of the catalytic subunit (hTERT) represents the limiting factor for telomerase activity. In transfection studies, high level of activity of hTERT promoter is found. whereas low copy numbers of hTERT mRNA are detected in vivo. To explain this discrepancy, a series of vectors containing the hTERT promoter and gene were transiently transfected into HeLa cells. Four important regions were identified. First, the core promoter has bidirectional activity. Second, the distal upstream region (-1821 to -811 bp) involved in the splicing of the first intron and could be a key of splicing specificity. Third, the intermediate promoter region (-800 to -300 bp) could play an important role in silencing the reverse promoter activity. Fourth, the structural gene (up to +1077) strongly reduced hTERT promoter activity. These results provide the first evidence that the first two exons play a major role in the down-regulation of the h TER T promoter in telomerase-positive cells. (C) 2002 Elsevier Science (USA). All rights reserved.