Telomerase activity is closely correlated with cellular proliferative activity in human tissues. Human cells with high proliferative potential, such as tumor cells or stem cells, exhibit telomerase activity, whereas most normal human somatic cells do not. Telomerase activity is tightly regulated by the expression of its catalytic subunit human telomerase reverse transcriptase (hTERT). Through an expression cloning approach, we identified E2F-1 as a repressor of the hTERT gene in human tumor cells. Ectopic expression of E2F-1 repressed hTERT promoter activity by inhibiting Sp1 activation of the hTERT promoter. In contrast to the repressor function of E2F-1 in human tumor cells, we demonstrated that E2F-1 is an activator of the hTERT gene in normal human somatic cells. Ectopically expressed E2F-1 activated the hTERT promoter through a noncanonical DNA binding site. E2F-1, E2F-2, and E2F-3 (but not E2F-4 and E2F-5) repressed hTERT promoter activity in human tumor cells, whereas they activated it in normal somatic cells. These contrasting effects of E2F transcription factors on the hTERT promoter could underlie the paradoxical biological activities of E2F, which can both promote and inhibit cellular proliferation and tumorigenesis.