This study was performed to determine upregulation of the human telomerase RNA component (hTR) and mRNA of the catalytic subunit of telomerase (hTERT) in (pre)malignant cervical lesions, to analyze possible intralesional heterogeneity of hTR expression, and to relate hTR and hTERT mRNA levels to telomerase activity levels and human papillomavirus (HPV) typing. hTR expression was determined by in situ hybridization (ISH) on paraffin-embedded sections, obtained from patients with cervical intraepithelial neoplasia (CIN) I-III or cervical cancer and from normal controls. hTR and hTERT mRNA expression were determined by semiquantitative rt-PCR on frozen samples from the same lesions. Data on telomerase activity and HPV were obtained from a previous study. hTR expression as determined by ISH was observed in 0 of 8 normal cervices, 1 of 14 CIN I, 15 of 28 CIN II, 21 of 30 CIN III, and 16 of 18 cervical cancer specimens. In general, hybridization patterns for hTR expression were homogeneous throughout the lesion. Frequency of hTR expression was related to grade of CIN/cervical cancer (P<.001). hTR expression, as determined by rt-PCR, was detected in 8 of 8 normal cervices, 2 of 2 CIN I, 12 of 14 CIN II, 23 of 23 CIN III, and 16 of 17 cervical cancer specimens. hTERT mRNA was detected in 1 of 8 normal cervices, 1 of 2 CIN I, 5 of 14 CIN II, 14 of 23 CIN III, and 11 of 17 cervical cancer specimens. hTR as determined by rt-PCR was not related to grade of CIN/cervical cancer, whereas hTERT mRNA expression was related to grade of CIN/cervical cancer (P<.01). hTR expression, as determined by ISH and hTERT mRNA expression by rt-PCR, were related to telomerase activity levels (P<.001, P<.05, respectively) and presence of oncogenic types of HPV (both P<.05). Our data show frequent upregulation of hTR and hTERT mRNA expression in CIN lesions, which appear to occur earlier than induction of telomerase activity. The fact that semiquantitative hTERT mRNA as well as hTR levels are related to telomerase activity levels illustrates that in (pre)malignant cervical lesions upregulation of both telomerase components may be important for functional telomerase.