Telomerase activity appears to be associated with cell immortalization and malignant progression. Understanding how telomerase activity is regulated in vivo is important not only for understanding the molecular biology of telomerase but also for the potential clinical application of anticancer drugs. This study evaluated telomerase activity and quantified the expression of human telomerase reverse transcriptase (hTERT) mRNA and human telomerase RNA (hTR) using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method before and after the exposure of cisplatin and 5-fluorouracil (5-FU) in two head and neck squamous cell carcinoma (HNSCC) cell lines. Two human HNSCC cell lines (PNUH-12 and SNU-899) were studied. Cell cytotoxicity, the change of telomerase activity, and hTERT mRNA and hTR expression by 5-FU and cisplatin exposure were assessed by MTT assay, TRAP assay, and real-time RT-PCR, respectively. In two cell lines, after cisplatin exposure, the telomerase activity and hTERT mRNA expression decreased, but hTR expression in- creased according to the concentration of drug. However, in both cell lines, the telomerase activity and hTR did not show any significant change after 5-FU treatment, but the expression of hTERT mRNA decreased. These results suggest that there may be other important regulating mechanism except hTERT mRNA as the regulation factor of telomerase activity in HNSCC cell lines.