The human telomerase reverse transcriptase (TERT) gene is transcriptionally inactivated in most differentiated cells but is reactivated in the majority of cancer cells. To elucidate how TERT is inactivated during differentiation, we applied all-trans retinoic acid (ATRA) to induce the differentiation of human teratocarcinoma (HT) cells and human acute myeloid leukemia (HL60) cells. We first showed that TERT promoter activity decreased rapidly, which preceded a gradual loss of endogenous telomerase activity following ATRA induction. To elucidate the underlying mechanisms of the reduced TERT promoter activity during differentiation, we performed epigenetic studies on the TERT promoter and found a progressive histone hypoacetylation coupled with a gradual accumulation of methylated cytosines in the TERT promoter. We also observed that the TERT promoter was less methylated in pluripotent HT cells than in multipotent HL60 cells throughout a 12-day differentiation process. This origin-dependent epigenetic change was also confirmed in histone acetylation studies, indicating that the TERT promoter was more resistant to deacetylation in HT cells than in HL60 cells. Taken together, our results demonstrate synergistic involvement of DNA methylation and histone deacetylation in the down-regulation of TERT promoter activity that may be dependent on the origin of the cell types, and they add new insight into the way telomerase activity may be regulated during differentiation. © 2004 Wiley-Liss, Inc.