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Immortal human cells maintain their telomeres by two independent mechanisms, a prevalent one dependent on de novo synthesis of telomeric DNA by telomerase, and a rarer one based on telomere recombination [alternative lengthening of telomeres (ALT)]. Studies with yeast have indicated that expression of telomerase inhibits telomere recombination. In the present study, we have investigated whether expression of telomerase in cells that use ALT would similarly reveal dominance of telomere elongation by telomerase over telomere recombination. Telomerase-negative WI38 VA13/2RA ALT cells were reconstituted for telomerase activity through ectopic expression of the enzyme subunits, hTERT and hTR, and the presence and function of telomerase and ALT were monitored during long term cell growth by enzymatic assays, detection of the ALT-associated PML bodies (APBs) and analysis of telomere dynamics. Our results indicate that telomerase activity and APBs persisted in the cells over at least 90 population doublings. The activity of both pathways on telomeres was determined by analysis of telomere length versus time by gel electrophoresis and in situ hybridization. ALT cells are characterized by very heterogeneous telomeres with a much longer average size than the telomeres of telomerase-positive cells. Telomere dynamics in our cells were compatible with both ALT and telomerase being biologically active since the long telomeres typical of ALT were maintained, while short telomeres, thought to be the preferential substrate of telomerase, were elongated. These findings, indicating that human cells may be capable of concomitantly utilizing both mechanisms of telomere maintenance without effects on their growth and viability, have implications for cancer therapy.