Telomerase is an attractive molecular target toward which to direct cancer therapeutic agents because telomerase activity is present in most malignant cells but undetectable in most normal somatic cells. Short duplex RNA (short-interfering RNA or siRNA) has recently been shown to be an effective method for inhibiting the expression of a given gene in human cells. Accordingly, we evaluated the ability of siRNA to inhibit telomerase activity in human cancer cells. Human cancer cell lines were transfected with 21 nt double-stranded RNA homologous to either the catalytic subunit of telomerase (human telomerase reverse transcriptase) or to its template RNA [human telomerase RNA(hTR)]. Both types of agents reduced telomerase activity in a variety of human cancer cell lines representing both carcinomas and sarcomas. Inhibition was dose-dependent, although modest in degree and, as expected, transient in duration. Transfection of HeLa cells using a plasmid containing the hTR gene in both forward and reverse orientations, intended to create a duplex of the hTR transcripts endogenously, resulted in decreased telomerase activity, decreased telomerase RNA content, and decreased telomeric DNA content but no decrease in the untargeted human telomerase reverse transcriptase mRNA. Telomerase inhibition by siRNA is notable because telomerase is regarded as restricted to the nucleus, whereas RNA interference is commonly regarded as restricted to the cytoplasm.