The enzyme telomerase is implicated in cellular resistance to apoptosis, but the mechanism for this resistance remains to be elucidated. The ability of telomerase to synthesize new DNA at telomeres suggests that this enzyme might function in the repair of double-stranded DNA breaks. To distinguish the effects of double-stranded DNA break damage and apoptosis on human telomerase activity, we treated the HL-60 human hematopoietic cancer cell line with clinical doses of the chemotherapeutic drug etoposide (0.5 to 5 microM), which allowed us to distinguish between events associated with DNA damage-induced cell cycle arrest, and events associated with apoptosis. Large (three- to seven-fold) upregulation of telomerase activity occurred soon after etoposide treatment (3 h) in S/G2/M-arresting populations; this upregulation was abolished at onset of apoptotic cell death. No upregulation of telomerase activity was observed in cells treated with a larger dose of etoposide (5 microM) that caused cells to undergo rapid apoptosis without intervening cell cycle arrests. These observations are consistent with a possible role for telomerase upregulation during the DNA damage response.