Based on the finding that telomerase is reactivated solely in cancer cells, the human telomerase reverse transcriptase (hTERT) promoter has recently been used to target cancer cells by gene therapy. The recent, surprising observation that telomerase is physiologically activated even in normal somatic cells during S-phase has raised concerns as to the safety of this methodology. To clarify this issue, the present study carefully examined the changes in endogenous telomerase activities, hTERT mRNA expression, and hTERT promoter-based transgene expression in normal and cancer cells at synchronized phases of the cell cycle. Telomerase activity and hTERT expression were detected at variable, but relatively high, levels in all 12 cancer cell lines, while both were undetectable in the 11 normal cell lines. In HepG2 cancer cells, the highest levels of hTERT expression and telomerase activity, seen in the G(1)/S- and S-phases, were 2-3-fold higher than the lowest levels of both, observed in G(0)-phase and during asynchronization. No hTERT expression or telomerase activitiy could be detected in normal WI-38 fibroblasts at any phase of the cell cycle, including S-phase. Consequently, activity of the shorter hTERT promoter, which was transferred into HepG2 cancer cells via adenovirus transduction, was stronger than that of the longer hTERT promoter at all phases and that of two representatives of ubiquitously strong promoters, at both S-phase and asynchronization, but not at G(0)-phase. In contrast, neither of hTERT promoters induced detectable transgene expressions in normal WI-38 cells at any cell cycle phase, including S-phase. These results, particularly the lack of problematic levels of S-phase-specific activation of hTERT promoters in normal cells, have promising implications for hTERT promoter-based targeted gene therapy of cancer.