The possibility of differentiating Brucella species and biovars by Southern blot hybridization of agarose gel-electrophoresed HindIII-digested genomic DNA with membrane protein- or cytoplasmic protein-specific gene probes was investigated on 92 reference and field strains representative of all known species and biovars. Based on the RFLP pattern observed, three gene probes, i.e. br25, 39ugpa and omp16 coding for membrane or cytoplasmic proteins differentiated B. melitensis, B. ovis and B. suis biovar 2 strains from each other and from the other Brucella species and biovars. Thus, the use of these specific gene probes could contribute, in addition to previously identified species- or biovar-specific markers, to the molecular identification and typing of Brucella isolates.