To determine the prevalence of serotypes of Streptococcus pneumoniae responsible for pneumonia with pleural effusion, we determined the capsular polysaccharide (PS) type directly on 49 pleural fluid specimens collected from pediatric patients during 2007 to 2009 with laboratory-confirmed pneumococcal pneumonia by using monoclonal antibodies and a multiplex, bead array immunoassay. Because the fluids had to be heated to remove nonspecific reactivity before being tested in the immunoassay and type 19A PS is heat labile, the pleural fluid samples were also tested for serotype 19A capsule gene locus by PCR. Use of the multiplex immunoassay combined with type-specific 19A PCR allowed for serotype determination on 40 of 49 pleural fluids. Pneumococcal pneumonia with pleural effusion was associated with a limited number of serotypes, with types 1, 3, 7F/A, and 19A accounting for 75% of the typeable cases. The concentration of capsular PS in the pleural fluids was often greater than 1 μg/ml and sufficient to inhibit the opsonic capacity of sera from individuals who had received the 23-valent pneumococcal PS vaccine. Based on the serotypes observed before and after introduction of the 7-valent pneumococcal conjugate vaccine, the recently licensed 13-valent pneumococcal conjugate vaccine may reduce the incidence of pneumonia with pleural effusions. Copyright © 2011, American Society for Microbiology. All Rights Reserved.