This study examined the role of antibodies against the X-protein, a surface-localized antigen frequently associated with streptococci causing mastitis in cattle, in the opsonization and phagocytosis of unencapsulated Streptococcus agalactiae. The analysis of various strains of serotype NT/X by flow cytometry, after labeling with a monoclonal antibody to X-protein, revealed that they consisted of a mixture of unstained and stained bacteria. Cloning of mother strains yielded clones of unstained bacteria but not homogeneous clones of stained bacteria. Analysis by ELISA of an unstained clone (4.1) derived from the reference NT/X strain 24/60 indicated that it expressed low amount of X-protein at its surface, about 25 times less than the stained clone 24/60 5.6. Colloidal gold immunolabeling showed the X-protein at the periphery of bacteria (of clone 5.6 and in lower amount of clone 4.1), at a distance from the cell wall. Bovine antibodies (essentially IgG) to X-protein behaved like the monoclonal antibody in the cytometric assay. They activated the classical pathway of complement as shown by the deposition of C1q and C4 on bacteria. Deposition of C4 also occurred on the low-surface-producing clone 4.1 in the presence of antibodies to X-protein, although less efficiently than on the high-surface-producing clone 5.6. When used alone, antibodies promoted the ingestion of bacteria and heat-inactivated immune serum promoted the chemiluminescence activity and the killing by polymorphonuclear cells. In conclusion, antibodies to X-protein induced the deposition of C3 by the classical pathway and were also able to stimulate opsonophagocytic killing of X-bearing S. agalactiae in the absence of deposited C3.