The capsular polysaccharide of Neisseria meningitidis serogroup W135 is expressed in both O-acetyl-positive (OA+) and O-acetyl-negative (OA-) forms. This study investigates the impact of OA status (OA+ versus OA-) on serological measurements of anti-W135 immunoglobulin G (IgG) antibodies in immunized adults. W135-specific serum antibody assignments were made for 28 postimmunization sera from adults by enzyme-linked immunosorbent assay using the meningococcal standard reference serum CDC1992. The established IgG concentration in micrograms per milliliter ([IgG]microg/ml) for CDC1992 against OA+ antigen (16.2 microg/ml) was used as a reference to assign a concentration of 10.13 microg/ml IgG against OA- antigen by cross-standardization. Overall, the IgG assignments for these sera were higher against OA+ antigen (geometric mean concentration [GMC] = 7.16 microg/ml) than against OA- antigen (GMC = 2.84 microg/ml). However, seven sera showed higher specific [IgG]microg/ml values against the OA+ antigen than against the OA- antigen. These sera were also distinguished by the inability of fluid-phase OA- antigen to compete for antibody binding to OA+ solid-phase antigen. Although there was no overall difference in functional activity measured by complement-mediated serum bactericidal assay (SBA) against OA+ and OA- target bacteria (geometric mean titers of 9,642 and 9,045, respectively), three serum specimens showed a large difference in SBA antibody titers against OA+ versus OA- W135 target bacteria, which may reflect different epitope specificities for these sera. Our data indicate that, for some sera, the agreement in anti-OA+ versus anti-OA- W135 IgG assignments is serum specific and does not reflect the functional (killing) activity in vitro.