Telomerase is a regulated enzyme and its activity is tightly associated with cell proliferation. The mechanisms of this association are unclear, but specific growth factors may regulate telomerase activity. The present study examines the effect of epidermal growth factor (EGF) on telomerase activity and identifies the signal transduction pathway involved in this process. EGF up-regulated telomerase activity in EGF receptor-positive cells after the activation of telomerase reverse transcriptase (TERT) mRNA expression. This activation was rapid, peaked after 6 or 12 h and was not blocked by the concurrent exposure to cycloheximide, suggesting a direct effect of EGF on TERT transcription. Transient expression assays revealed that EGF activates the hTERT promoter and that the proximal core promoter is responsible for this regulation. The activation of hTERT mRNA expression by EGF was specifically blocked by MEK inhibitor, and in vitro kinase assays demonstrated that ERK is activated in response to EGF. Transient expression assays using mutant reporter plasmids revealed that an ETS motif located in the core promoter of hTERT is required for the EGF-induced transactivation of hTERT. Overexpression of wild type Ets in cells enhanced the EGF effect on hTERT transcription, while that of dominant negative Ets significantly repressed EGF action. These findings suggest that EGF activates telomerase through the direct activation of TERT transcription, in which the Ras/MEK/ERK pathway and Ets factor play major roles. Our data support the notion that growth factors directly regulate telomerase via specific signal transduction pathways.