Human telomerase reverse transcriptase (hTERT) mRNA expression seems to play an important role in cervical carcinogenesis. Analysis of the hTERT promoter region revealed the presence of a CpG island and a high overall GC content, suggesting a possible role for methylation in the regulation of hTERT gene expression. The present study was designed to evaluate the role of hTERT promoter methylation and E6/E7 human papilloma virus 16 (HPV-16) mRNA expression in hTERT regulation in premalignant cervical specimens. The methylation status of the hTERT promoter gene and hTERT mRNA quantification were investigated in 26 normal and 64 specimens of abnormal cytology using the MethyLight technique, Telo-TAGGG hTERT Quantification Kit and LightCycler technology. E6/E7 HPV-16 mRNA expression was also evaluated. No significant correlations were observed between hTERT mRNA expression and hTERT promoter methylation, as well as between telomerase activity and hTERT promoter methylation in normal and in premalignant cervical specimens. E6/E7 HPV-16 mRNA expression was observed in 72% of HPV-16-infected samples and was correlated with hTERT mRNA expression and telomerase activity (P < 0.05). This is the first study investigating the role of hTERT promoter methylation in hTERT mRNA expression and telomerase activity in premalignant lesions. The observed lack of correlation suggests that other mechanisms might be involved in the regulation of hTERT expression. The correlation between hTERT mRNA and E6/E7 mRNA expression confirms the role of HPV infection in hTERT regulation.