The p16 and p53 tumor suppressor proteins, which are frequently altered in malignant gliomas, have been noted as regulators of telomerase activity. However, the link between telomerase regulation and these suppressor proteins has not been adequately clarified. In the present study, we demonstrated that p16, as well as p53, suppress telomerase activity through transcriptional regulation of human telomerase reverse transcriptase (hTERT) in malignant glioma. To examine the effect of p16 and p53 on telomerase activity, we utilized wild-type p16 or p53 expression plasmid and three human glioma cell lines differing in their p53 and p16 status. Restoring p16 significantly reduced the level of telomerase activity of glioma cells. Furthermore, cotransfection of the p16 gene with 5'-deletion constructs of the hTERT promoter carrying Sp1 binding sites, repressed the transcriptional activity of hTERT promoter in p16-deleted cells. In addition, electrophoretic mobility shift assay revealed that p16 expression inhibited the binding of Sp1 to the consensus Sp1 responsive element, indicating that the recruitment of Sp1 to the hTERT proximal core promoter is inhibited by p16 protein. These results were similar to those from a p53 transfection study in p53-mutated cells. These findings implicate p16 in the transcriptional regulation of telomerase activity by inhibiting the function of Sp1 in human malignant gliomas.