Telomerase, a ribonucleoprotein complex is activated in the vast majority of human malignancies, including prostate cancer. Its inhibition is a putative way to affect cancer proliferation and might be used in the therapy of tumors. We analysed the influence of antisense phosphorothioate oligonucleotides (PTO) against the reverse transcriptase subunit of telomerase on prostate cancer cell viability, telomerase activity and telomere length. DU145 prostate cancer cells were cultivated in PTO containing medium. The PTO-incorporation was confirmed by confocal laser scanning microscopy. Cell viability was measured by a WST-1 tetrazolium assay. After 15 days of antisense PTO treatment, a significant inhibition of cell viability occurred. Telomerase activity was determined by a telomeric repeat amplification protocol (TRAP) assay and telomere length by Southern blot analysis. Since the long-term telomerase antisense treatment reduces the viability of prostate cancer cells significantly, this antisense approach could be a new therapeutic strategy to treat patients with advanced prostate cancer.