A number of different approaches have been developed to inhibit telomerase activity in human cancer cells. In this study, the effect of antisense oligonucleotides (ODNs) by targeting human telomerase reverse transcriptase (hTERT) mRNA in a laryngeal cancer cell line (Hep-2) was investigated. A 20mer antisense oligodeoxynucleotide targeting the most open part of hTERT mRNA (anti-hTERT) and a mismatched control sequence were synthesized. Cells were treated daily with oligonucleotides for up to 72 hours. hTERT mRNA expression was measured by the reverse transcription polymerase chain reaction (RT-PCR) assay; telomerase activity by the telomerase PCR ELISA assay kit (TRAP; Boehringer Mannheim, GmbH, Mannheim, Germany). Cell viability after administration of ODNs was determined using the MTT assay. Morphological changes were examined by haematoxylin and eosin staining. The cell cycle was analyzed using flow cytometry. It was found that antisense treatment induced a decrease in hTERT mRNA expression, telomerase activity, cell growth rate, cell viability, and an increase in apoptosis. The results suggest that inhibition of telomerase activity in Hep-2 cells by short-term antisense treatment against the mRNA of hTERT results in apoptotic cell death. The treatment with anti-hTERT may be useful as a treatment modality for laryngeal squamous carcinoma.