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To produce monoclonal antibodies (MAbs), Balb/c AnN Crl BR mice were inoculated with the cell suspension of a Hungarian Mycoplasma bovis strain designated 26034. Three days after the last immunization the spleen of the immunized mouse was removed aseptically. The fusion of spleen cells with Sp2/0-Ag14 murine myeloma cells was performed in the presence of polyethylene glycol. The obtained hybrid cells were selected with hipoxantine, aminopterine and thymidine (HAT) medium. Two weeks after the fusion, the supernatants of the grown cells were tested by a self-developed indirect enzyme-linked immunosorbent assay (ELISA). The results showed that 63 antibody-producing hybridomas had been obtained. For accurate determination of the molecular weight of antigen determinants, the supernatants giving positive reaction in the ELISA were tested by Western blotting. According to the results, the obtained MAbs recognize the antigen determinants of the following molecular weights: 1B11: 63 kDa, 1C7: 63 kDa, 2C5: 22, 25 and 27 kDa, 2C9: 69 kDa, 3G12: 67, 69 and 72 kDa, 4H9: 63 kDa, 5B8: 22, 25 and 27 kDa, 5D3: 22, 25 and 27 kDa, 5C11: 69 kDa, 5E5: 22, 25 and 27 kDa, 6F11: 63 kDa, and 6H10: 22, 25 and 27 kDa. The 12 cell groups selected on the basis of the Western blotting were cloned twice by end-point dilution method. The cloned cells were propagated, and with 5 cell lines antibodies were produced in the CELLine bioreactor (Integra Biosciences, Zurich, Switzerland). Cell line 3G12 showed the highest productivity with an average daily output of 1.5 mg immunoglobulin. Cell line 5E5 produced 1.1 mg, 6H10 0.8 mg, 2C9 0.47 mg and 6F11 0.4 mg antibody per day. The isotype of the antibodies was determined by ELISA. The antibodies produced by the 12 cell lines tested were assigned to the IgG1 subclass according to the heavy chain. Ten cell lines produced κ and two produced λ light-chain antibody. Possible cross-reactions of the produced monoclonal anti-M. bovis antibodies with certain Mycoplasma, Ureaplasma and Acholeplasma species were tested by an indirect ELISA procedure. All of the 12 antibodies tested gave a reaction with the antigen of M. bovis strain designated 26034. MAbs 3G12 (67, 69, and 72 kDa) and 5B8 (22, 25, and 27 kDa) gave no cross-reaction with antigens other than strains of the homologous Mycoplasma species. The other antibodies reacted with the M. bovigenitalium F7, M. anseris 8389, M. oculi, and M. gallisepticum s6 Holland antigens. Owing to its high specificity and affinity, the antibody produced by the cell line 3G12 is primarily considered suitable for use in immunodiagnostic tests of M. bovis infections.