The replicative potential of human CD56+ CD3− natural killer (NK) cells is unknown. We found that by exposing NK cells to the leukaemic cell line K562 genetically modified to express 4-1BB ligand and interleukin 15 (K562-mb15-41BBL), they expanded for up to 30 population doublings, achieving numbers that ranged from 1·6 × 105 to 1·2 × 1011% (median, 5·9 × 106%; n = 7) of those originally seeded. However, NK cells eventually became unresponsive to stimulation and died. Their demise could be suppressed by enforcing the expression of the human telomerase reverse transcriptase gene (TERT). TERT-overexpressing NK cells continued to proliferate in response to K562-mb15-41BBL stimulation for more than 1 year of culture, while maintaining a normal karyotype and genotype. Long-lived NK cells had high cytotoxicity against myeloid and T-lineage leukaemic cells. They remained susceptible to genetic manipulation, becoming highly cytotoxic to B-lineage leukaemic cells after expression of anti-CD19 signaling receptors. Thus, human NK cells have a replicative potential similar to that of T lymphocytes and their lifespan can be significantly prolonged by an increase in TERT activity. We suggest that the methods described here should have many applications in studies of NK cell biology and NK cell-based therapies.