Telomeres are usually maintained about an equilibrium length, and the set point for this equilibrium differs between species and between strains of a given species. To examine the requirement for telomerase in mediating establishment of a new telomere length equilibrium, we generated interspecies crosses with telomerase mTR knockout mice. In crosses between C57BL/6J (B6) and either of two unrelated mouse species, CAST/Ei and SPRET/Ei, telomerase mediated establishment of a new telomere length equilibrium in wild-type mTR(+/+) mice. This new equilibrium was characterized by elongation of the short telomeres of CAST/Ei or SPRET/Ei origin. In contrast, mTR(-/-) offspring of interspecies crosses failed to elongate telomeres. Unexpectedly, haploinsufficiency was observed in mTR(+/-) heterozygous interspecies mice, which had an impaired ability to elongate short SPRET/Ei or CAST/Ei telomeres to the new equilibrium set point that was achieved in wild-type mTR(+/+) mice. These results demonstrate that elongation of telomeres to a new telomere set point requires telomerase and indicate that telomerase RNA may be limiting in vivo.