We determined telomerase activity semiquantitatively and in-cell telomerase activity in exfoliated urinary cells obtained from urological neoplasias. Semiquantitative determination of telomerase activity was performed using a fluorescent-based telomeric repeat amplification protocol (TRAP) and telomerase activity at the cellular level was determined by an in situ TRAP assay. Fluorescent-based TRAP assay detected urinary telomerase activity in about 80% patients with urological neoplasia before treatment, whereas urinary cells obtained from 30% patients after/during treatment with either chemotherapy or operation (class II-V) had detectable telomerase activity. In contrast, the in situ TRAP assay detected telomerase-positive cells in 85% patients before treatment and 60% patients after/during treatment. Of note is a dissociation of the results between fluorescent-based TRAP assay and that of in situ TRAP assay in some patients. Some patients without detectable telomerase activity using the fluorescent-based TRAP assay showed a low frequency of telomerase-positive cells in the urine. A combination of semiquantitative analysis and an in situ TRAP assay to detect telomerase-positive cells might be a useful tool in the detection and monitoring of patients with urological neoplasias.