A novel pentacyclic acridine, 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4), has been identified as a potent inhibitor of telomerase in the cell-free telomeric repeat amplification protocol (TRAP). Modeling and biophysical studies suggest that RHPS4 inhibits telomerase through stabilization of four-stranded G-quadruplex structures formed by single-stranded telomeric DNA. In contrast to G-quadruplex interactive telomerase inhibitors described previously, RHPS4 inhibited telomerase at submicromolar levels (50% inhibition in the TRAP assay at 0.33 ± 0.13 μM). Moreover, RHPS4 exhibited a wide differential between this potent inhibition of telomerase and acute cellular cytotoxicity (mean IC₅₀ value of 7.02 μM in 4-day growth inhibition assay). RHPS4, when added to 21NT breast cancer cells at nonacute cytotoxic concentrations (200 nM) every 3 to 4 days, induced a marked cessation in cell growth after 15 days. Similar effects were observed using another cell line possessing relatively short telomeres, A431 human vulval carcinoma cells, but not in a human ovarian carcinoma cell line (SKOV-3) possessing relatively long telomeres. In 21NT cells, growth cessation was accompanied by an increase in cells in the G₂/M phase of the cell cycle, a reduction in cellular telomerase activity, and a lower expression of the hTERT gene. These effects occurred in the absence of a detectable reduction in telomere length as measured by slot blotting. RHPS4 also induced a cessation of growth of GM847 cells that maintain telomeres by a nontelomerase alternative mechanism for lengthening telomeres (ALT) after 15 days. RHPS4 represents a promising G-quadruplex interactive small molecule that is a potent cell-free inhibitor of human telomerase and induces growth inhibitory effects in human tumor cell lines after prolonged (2-week) exposure to nonacute cytotoxic drug concentrations.