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Telomere length maintenance is essential for cellular immortalization, and thus tumorigenesis. Most human tumors and immortal cell lines maintain their telomeric DNA via the activity of a specialized reverse transcriptase, telomerase. Stabilization of telomeric repeat tracts may also be achieved through a telomerase-independent mechanism, referred to as alternative lengthening of telomeres (ALT). ALT cells are telomerase negative and are characterized by extremely long and heterogeneously sized telomeres and novel multiprotein structures called ALT-associated PML nuclear bodies which are unique to ALT cells. To determine if reconstitution of telomerase activity suppressed ALT and restored wild-type telomere lengths, we introduced the catalytic subunit of telomerase into two ALT cell lines. Initially, two clonal lines exhibited enrichment of shorter telomeres while maintaining a population of ultra-long telomeres similar to that observed in the parental line, suggesting that telomerase is stabilizing the shorter telomeres in the population. Telomere length in the third clonal line was not detectably different from that in the parental cell line. One clonal line with a phenotype of shorter telomeres maintained this pattern over time in culture while the second gradually reverted to the parental ALT telomere length pattern, concurrent with reduction of telomerase activity. All clones continued to maintain ALT-associated PML nuclear bodies regardless of whether telomerase was present. The data suggest that introduction of telomerase activity alone is not sufficient to completely repress ALT, that telomerase acts preferentially on the shortest telomeres in the culture and that the ALT and telomerase pathways may be present concurrently in mammalian cells.