Abstract Our long-term goal is to determine the mechanisms of telomerase regulation during development. The hTERT gene, encoding the limiting subunit of human telomerase, is primarily regulated at the level of transcription. It is highly expressed in pluripotent stem cells, but stringently repressed in most somatic cells. Regulation of transcription during development and differentiation often involves distal elements and chromatin reorganization, in addition to proximal promoter elements. We previously showed that the endogenous hTERT locus was embedded in a condensed chromatin domain in many somatic cells. Consistent with the vital role of chromatin in its tight regulation, we found that an episomal hTERT locus in human fibroblasts was not subjected to repression, whereas a chromosomally integrated hTERT locus recapitulated its native regulation. To study the mechanisms of hTERT repression, we developed a novel technical platform, the recombinase-mediated BAC targeting or RMBT method, for targeted integration of single-copy BAC reporters into specified chromosomal sites. This technique enables us to study the regulation of hTERT gene in its native genomic context. Here, we hypothesize that 1) the interplay between distal elements and core promoter in their native chromatin contexts is important for hTERT repression in somatic cells and reactivation in cancer cells; and 2) polymorphic regulatory elements of the hTERT gene impact human aging and tumorigenesis. We propose three specific aims: 1) determine the recruitment of corepressor complexes and their roles in hTERT repression; 2) determine the regulation of hTERT promoter by Ets family proteins in normal and cancer cells; 3) determine the function and genetic variation of a polymorphic element in hTERT regulation and human longevity.