Brucella (B.) species lack classical virulence factors, but escape effectively the immune response of the host. The species Brucella abortus and Brucella melitensis infect predominantly cattle and small ruminants such as sheep or goats, respectively, but account also for most human cases. These two species share remarkably similar genomes but different proteomes have been demonstrated. This might be one of the reasons for their host specificity. A comprehensive identification of immunodominant proteins of these two species using antibodies present in the serum of naturally infected ruminants might provide insight on the mechanism of their infection in different hosts. In the present study, whole-cell protein extracts of B. abortus and B. melitensis were separated using SDS–PAGE and western blotting was performed using field sera from cows, buffaloes, sheep and goats. Protein bands that matched with western blot signals were excised, digested with trypsin and subjected to protein identification using MALDI-TOF MS. Identified proteins included heat shock proteins, enzymes, binding proteins and hypothetical proteins. Antibodies against the same set of antigen were found for all species investigated, except for superoxide dismutase of B. melitensis for which antibodies were demonstrated only in sheep serum. Brucellae appear to express these proteins mainly for their survival in the host system during infection.