In vivo immunization, fusion, antibody detection, and cryopreservation procedures for monoclonal antibody production against antigens of Brucella abortus are described. Splenocytes from BALB/c mice immunized with irradiated B. abortus S2308 were fused with Sp2/O-Ag14 myeloma cells and 61 hybridomas secreting anti-Brucella antibodies were cloned. Hybridoma antibody synthesis was detected effectively and most efficiently by enzyme-linked immunosorbent assays. Antibodies from clones of hybridoma A23 reacted with S19 and S2308 whole bacterial cells, while hybridoma B49 reacted primarily with alkali--treated lipopolysaccharides of S19, S1119.3 and S2308. Cryopreservation of clones had no major effect on antibody synthesis. The application of monoclonal anti-Brucella antibodies in the differential diagnosis of bovine brucellosis is discussed.