The brucellae are Gram-negative bacteria that cause an important zoonosis. Studies with the main Brucella species have shown that the O-antigens of the Brucella smooth lipopolysaccharide are α-(1 → 2) and α-(1 → 3)-linked N-formyl-perosamine polysaccharides that carry M, A and C (A = M, A>M and A<M) epitopes relevant in serodiagnosis and typing. We report that, in contrast to the B. suis biovar 1 O-antigen used as a reference or to all described Brucella O-antigens, B. suis biovar 2 O-antigen failed to bind monoclonal antibodies of C (A = M), C (M>A) and M specificities. However, the biovar 2 O-antigen bound monoclonal antibodies to the Brucella A epitope, and to the C/Y epitope shared by brucellae and Yersinia enterocolitica O:9, a bacterium that carries an N-formyl-perosamine O-antigen in exclusively α-(1 → 2)-linkages. By (13)C NMR spectroscopy, B. suis biovar 1 but not B. suis biovar 2 or Y. enterocolitica O:9 polysaccharide showed the signal characteristic of α-(1 → 3)-linked N-formyl-perosamine, indicating that biovar 2 may altogether lack this linkage. Taken together, the NMR spectroscopy and monoclonal antibody analyses strongly suggest a role for α-(1 → 3)-linked N-formyl-perosamine in the C (A = M) and C (M>A) epitopes. Moreover, they indicate that B. suis biovar 2 O-antigen lacks some lipopolysaccharide epitopes previously thought to be present in all smooth brucellae, thus representing a new brucella serovar that is M-negative, C-negative. Serologically and structurally this new serovar is more similar to Y. enterocolitica O:9 than to other brucellae.