We investigated the diagnostic significance of telomerase activity in gynecological malignancies. Tissue samples were obtained from 24 cervical cancers, 27 uterine cancers (22 endometrial cancers and five sarcomas), 33 ovarian cancers (31 epithelial tumors and 2 germ cell tumors), and 11 benign ovarian tumors. In addition, cervical cytology specimens were obtained from 30 squamous intraepithelial lesions (13 low grade and 17 high grade), and from 22 normal females. Telomerase activity was detected using the TRAP assay, and the relative telomerase activity was obtained using the BioMax DNA image analysis system. Telomerase activity was detected in 22/24 (91.7%) cervical cancers, 23/27 (85.2%) uterine tumors and 30/33 (90.9%) ovarian cancers. Weak telomerase activity was detected in two mature cystic teratomas and also found in 9/17 (52.9%) high grade SIL and 2/13 (15.4%) low grade lesions. Telomerase activity showed no relationship with tumor histology or clinical stage, and there was no statistically significant difference between patients with uterine cancer and ovarian cancer. Relative telomerase activity showed a correlation with the dilution assay, and significantly higher telomerase activity was found in uterine cervical cancer compared with precancerous lesions and in ovarian cancer compared with benign ovarian tumors. After establishment of an assay for telomerase, it may be useful for cancer diagnosis and identification of high-risk groups.