Telomere shortening controls the entry of cells into senescence. Functional expression of the telomerase catalytic subunit (human telomerase reverse transcriptase or hTERT) stabilizes telomere length and extends the life span of various normal human cells. Our aim was to assess the role of telomerase activity and telomere maintenance in regulating the proliferation of activated human hepatic stellate cells (HSCs), to establish an immortal human HSC cell line. Human HSCs were isolated from surgical specimens of normal liver and infected with a retrovirus expressing hTERT. Ectopic expression of hTERT reconstituted telomerase activity and maintained telomere length in human HSCs. Control human HSCs, which were either not infected or infected with a retroviral vector containing only the neomycin resistance gene, showed no detectable telomerase activity and had slightly shortened telomeres. These telomerase-negative HSCs entered a nondividing state after about 9 to 15 passages and senesced. In contrast, telomerase-positive HSCs to date have undergone 69 passages. Telomerase-positive HSCs did not undergo oncogenic transformation and exhibit morphologic and functional characteristics of activated HSCs. Microarray and RT-PCR analysis showed that mRNA expression patterns in telomerase-positive HSCs are very similar to those in activated human HSCs. Plating telomerase-positive HSCs on a basement membrane-like matrix reverts them toward a more quiescent phenotype. In conclusion, introduction of hTERT into activated human HSCs immortalizes them and maintains their activated phenotype. This newly developed cell line will be a useful tool to study the cell biology of human HSCs in culture.