To develop monoclonal antibodies (MAbs) to neutralize human telomerase, an epitope (hTERT(7)) in reverse transcriptase domain of hTERT was synthesized and was used to immunize BALB/c mice. Hybridomas were generated and screened by enzyme-linked immunoadsorbent assay (ELISA) for specific MAbs. One hybridoma M2 clone, isotyped IgG(1), was established. The competitive assay confirmed that the M2 antibody was hTERT(7) specific, and the affinity constant was about 1 x 10(6) M(-1). M2 could recognize cell extracts from HeLa cancer cells but not those of normal 2BS cells in ELISA assay. For in situ staining immunohistochemically, the positive staining presented in the nuclear compartment of HeLa, while 2BS was nonreactive. In TRAP-PCR ELISA, M2 markedly decreased the activities of human telomerase in HeLa cells. The sequencing of M2 heavy chain variable region proved its mouse origin. The results demonstrated that the developed mouse MAb should be hTERT specific and could not only recognize native cellular hTERT in ELISA and immunohistochemistry, but also neutralize telomerase activities. Thus, it could be hoped that the antibody could be used in clinical diagnosis and treatment of cancers.