Telomerase activity plays an important role in the two complementary processes of cellular immortalization and senescence. This enzyme is active in almost all tumors, but also in inflammatory and many normal proliferating cells. Therefore, the main limits of molecular determinations, such as telomeric repeat amplification protocol assay is that they are not able to discriminate between the enzymatic activity of tumor and normal cells. The most appropriate technique for this would be immunohistochemical determination using monoclonal antibodies. Very few monoclonal antibodies (Mabs) directed against the human telomerase reverse transcriptase (hTERT) are commercially available and in the present study, we developed a new Mab directed against this protein (TERT-3 36-10) to investigate the possibility of detecting immunoreactivity to this Mab by immunohistochemical and flow cytometric approaches. Immunohistochemical determination showed a lack of reactivity to the Mab in highly differentiated striated muscle tissue, a variable reactivity in dysplastic cervical epithelial tissue and similar and widespread immunoreactivity in cell lines and clinical tumors. Furthermore, we demonstrated the ability of this Mab to inhibit enzyme activity in cell extract from MCR bladder tumor cell line.