In the present study, hybridomas were developed for the production of monoclonal antibodies (MAb) against recombinant 26-kDa periplasmic protein (rBP26) of Brucella abortus. A set of six stabilized hybridoma cell lines were generated. Monoclonal antibodies secreted by all of these clones exhibited reaction for rBP26, as well as with the protein of 26-kDa, derived from whole cell lysate of B. abortus 544. Three out of six MAbs were IgG1, two were IgM, and one was IgG2b in nature. These MAbs;; did not show any cross-reaction with whole cell lysate of Yersinia enterocolitica 0: 9, Vibrio cholerae, Salmonella typhimurium, and Escherichia coli O157 in Western blot analysis. Reactivity of these MAbs was further assessed with other organisms of Brucella species, namely, B. abortus 544, B. canis, B. melitensis 16M, and B. suis. Collectively, these data suggest that these MAbs may have the potential for their use in the detection or Brucella species with high specificity.