Seventy-seven Brucella reference and field strains from different geographic origins and hosts representing the six recognized species and their different biovars were analysed for diversity of their genes encoding the major 25 and 36 kDa outer-membrane proteins (OMPs) by PCR-RFLP. The 25 kDa OMP is encoded by a single gene (omp25) whereas two closely related genes (omp2a and omp2b) encode and potentially express the 36 kDa OMP. Analysis of PCR products of the omp25 gene digested with nine restriction enzymes revealed two species-specific markers, i.e. the absence of the EcoRV site in all Brucella melitensis strains and an approximately 50 bp deletion at the 3' terminal end of the gene in all Brucella ovis strains. Analysis of PCR products of the omp2a and omp2b genes digested with 13 restriction enzymes indicated a greater diversity than the omp25 gene among the six Brucella species and within the Brucella abortus, Brucella suis, B. melitensis and B. ovis species. Greater polymorphism was also detected for the omp2b than for the omp2a gene, especially in B. ovis which seemed to carry two similar (but not identical) copies of omp2a instead of one copy each of omp2a and omp2b for the other Brucella species as was previously suggested by Ficht et al. (1990; Mol Microbiol 4, 1135-1142). Results of PCR-RFLP indicated that distinction can be made between Brucellia species and some of their biovars, except between B. canis and B. suis bv. 3 and 4, on the basis of the size and diversity of their major OMP genes, and that it could be of importance for diagnostic, epidemiological and evolutionary study purposes.