Telomerase activity is not readily detected in resting human T lymphocytes, however upon antigen presentation, telomerase is transiently upregulated. Presently, it is not known if telomerase activation is necessary for the proliferation of T cells or for the maintenance of telomere lengths. In this study, we found that telomerase activation is not required for the short- term proliferation of T cells and that telomeres progressively shorten in a heterogeneous population of T cells, even if telomerase is detected. By measuring telomerase activity at the single-cell level using quantitative ddPCR techniques (ddTRAP) and by monitoring changes in the shortest telomeres with more sensitive telomere length measurement assays, we show that only a subset of CD28+ T-cells have robust telomerase activity upon stimulation and are capable of maintaining their telomere lengths during induced proliferation. The study of this T-cell subset may lead to a better understanding on how telomerase is regulated and functions in immune cells.