We have developed and characterized a rapid semiautomated pneumococcal serotyping system incorporating a pneumococcal lysate preparation protocol and a multiplex serotyping assay. The lysate preparation incorporates a bile solubility test to confirm pneumococcal identification that also enhances assay specificity. The multiplex serotyping assay consists of 24 assays specific for 36 serotypes: serotypes 1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/(33C), 11A/11D/11F, 12A/12B/12F, 14, 15B/(15C), 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F. The multiplex assay requires a flow cytometer, two sets of latex particles coated with pneumococcal polysaccharides, and serotype-specific antibodies. Fourteen newly developed monoclonal antibodies specific for common serotypes and a pool of polyclonal rabbit sera for some of the less-common serotypes are used. The two monoclonal antibodies specific for serotypes 18C and 23F recognize serotypespecific epitopes that have not been previously described. These monoclonal antibodies make the identification of the 14 common serotypes invariant. The specificity of the serotyping assay is fully characterized with pneumococci of all known (i.e., 90) serotypes. The assay is sensitive enough to use bacterial lysates diluted 20 fold. Our serotyping system can identify not only all the serotypes in pneumococcal vaccines but also most (>90%) of clinical isolates. This system should be very useful in serotyping clinical isolates for evaluating pneumococcal vaccine efficacy.Streptococcus pneumoniae is a well-known human pathogen and a major etiologic agent for pneumonia, meningitis, otitis media, and sepsis, primarily among young children and older adults (3). S. pneumoniae is divided into 90 serotypes, based on its expression of serologically distinct carbohydrate capsules (6). Antibodies to a capsular polysaccharide (PS) provide protection against pneumococci expressing the same capsular serotype. Currently available pneumococcal vaccines contain a mixture of capsular PS of multiple serotypes. One pneumococcal vaccine (called PS vaccine) contains capsular PS from 23 commonly found serotypes (17). The most recently developed type of vaccine (called conjugate vaccine) contains capsular PS from 7 to 11 serotypes that are conjugated to a protein molecule (26). A 7-valent conjugate vaccine was introduced in 2000 for routine immunization in the United States and has reduced the incidence of invasive pneumococcal diseases in children (25).Accurate, efficient serotyping of pneumococcal isolates is important for measuring the efficacy of pneumococcal vaccines. Since the vaccine-induced antibodies provide serotypespecific protection, following the introduction of a new pneumococcal vaccine, pneumococci expressing the serotypes included in the vaccine would become less common while the prevalence of the pneumococci expressing nonvaccine serotypes would stay the same. As a result, a reduction in vaccine serotypes reflects the vaccine efficacy. In some cases, pneumococci expressing the non...