Telomerase is a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends using an RNA component as a template. Extension of telomeric repeats by telomerase prevents telomere shortening with cell divisions and contributes to chromosomal stability, possibly leading to immortalization of the cells. In the present study, we determined the telomerase activity of gynecological tumors and cell lines using a newly developed non-radioisotope telomeric repeat amplification protocol. A total of 21 cell lines derived from cervical cancer, endometrial cancer, ovarian cancer, and choriocarcinoma was examined, and all lines were found to be positive for telomerase activity, although the activity varied among cell types. A total of 50 gynecological malignant tumors was also examined, and 10 of 12 (83%) cervical cancers, 12 of 13 (92%) endometrial cancers, 18 of 21 (86%) ovarian cancers, 2 of 2 tubal cancers, and 1 of 1 vulvar cancer were found to be positive for telomerase activity. A total of 88% of gynecological tumors tested was thus found to be telomerase positive. However, no significant correlation was observed between telomerase activity and clinical features for any tumor type, although ovarian tumors expressing high telomerase activity tended to be more invasive. In contrast to that in malignant tumors, telomerase expression was weak and less common in premalignant lesions, with 5 of 7 cervical intraepithelial lesions and 4 of 6 borderline ovarian tumors exhibiting faint activity. Nine benign uterine lesions were also examined, and all were negative for telomerase activity except 1 uterine myoma, which had a weak signal. Three benign ovarian cysts examined had weak telomerase activity. These findings suggest that telomerase activation is common in gynecological malignant tumors and may be a critical step in their pathogenesis. However, premalignant lesions and some types of benign tumors also express weak telomerase activity.