The role of O-acetyl group on binding characterization to pneumococcal type 9V polysaccharide (PS) was studied by using 9V PS monoclonal antibody (MAb) and rabbit antiserum. Alkali hydrolysis of O-acetyl groups from the 9V PS markedly decreased binding of rabbit 9V antiserum to the 9V PS. However, the binding with 9V MAb was less affected by the loss of O-acetyl content indicating that the MAb epitope is in the 9V backbone. As expected from the structure of the 9A PS the binding reaction with rabbit 9A antisera was independent of the O-acetyl content. The antibody binding of alkali-hydrolyzed 9V PS with 9V antisera was similar to the 9A antisera. The Factor g epitope reported by the Danish Serum Institute contained in the group 9 PS is considered to be the O-acetyl determinant. Thus, factor g-specific rabbit 9V antisera recognized an epitope that involves O-acetyl groups, and 9V MAb recognizes an epitope in the backbone structure, less affected by the O-acetyl group. Type 9V pneumococci were killed more effectively by human PMNs in the presence of the 9V PS MAb and complement compared to rabbit 9V antiserum. Passive immunity could provide effective protection for pneumococcal infection in early life.