Telomerase has been shown to be essential for unlimited cell proliferation and has been linked to immortality. However, still very little is known about the mechanism by which this enzyme is activated or inactivated. To investigate its regulation, we closely monitored telomerase activity during HL60 cell differentiation induced by either 1α,25-dihydroxyvitamin D₃orall-transretinoic acid. To that effect, we used a new combination of TRAP assay and SPA, which provides reproducible data for the quantitation and detection of variations in enzyme activity. We thereby observed that the decrease in telomerase activity after induction of differentiation by either of these agents is an early event of the differentiation process rather than its consequence, and that it is independent of the growth arrest pathway. It is neither due to a reduced expression of its RNA component nor to the appearance of a telomerase inhibitor in differentiating cells but is parallel to an increase in p21 and Rb mRNA expression.