Telomerase, a ribonucleoprotein that directs the synthesis of telomeric DNA repeats and compensates for the telomeric losses that occur with cell division, is absent from most mortal cells but is present in immortal cells. Telomerase activity is thought to be essential for continuous cell division as seen in malignant tumor cells, and its inhibition could be a strategy for anti-cancer therapy. We prepared a hammerhead ribozyme (teloRZ) directed against the RNA component of human telomerase and tried to find if it could serve as an inhibitor of telomerase. TeloRZ showed a specific cleavage activity against a synthesized portion of the telomerase RNA component used as the substrate. Furthermore, when added to cell extracts from HepG2 or Huh-7, human hepatocellular carcinoma derived lines, teloRZ inhibited the telomerase activity in both. These findings support the potential application of ribozymes capable of telomerase inhibition as new therapeutic agents directed against immortalized cancer cells.