Voided urine from patients with bladder cancer and from control patients with either hematuria or with no urologic conditions were examined for telomerase activity in order to explore the possibility that this activity could be used as a marker for the detection of bladder cancer. This assay was found to have an overall sensitivity in detecting bladder cancer of 85% (88/104) with 79% (23/29) grade 1 tumors, 84% (32/38) grade 2 tumors, 87.5% (28/32) grade 3 tumors, and 100% (5/5) carcinoma in situ testing positive. This compared favorably with urinary cytology which had an overall sensitivity of 51% and sensitivities of 13%, 44%, 82%, and 100% for grades 1, 2, 3 tumors and carcinoma in situ, respectively. The specificity of telomerase in patients with benign causes of hematuria was 66%, and in normal volunteers without urologic conditions, it was 100%. Assessment of nuclear matrix protein suggested comparable sensitivity and specificity. Evaluation of bladder tumor antigen showed less sensitivity for low-grade disease and less specificity, as it was influenced by inflammation and instrumentation. Telomerase thus seems to be a means whereby low-grade tumors may be detected in examination of voided urine and may offer an advantage in monitoring for recurrent disease.