Streptococcus pneumoniae is the most frequent cause of pneumonia and meningitis. Because S pneumoniae can colonize the upper respiratory tract and antibiotic treatment may inhibit growth, culture-based diagnosis can be problematic. An immunohistochemical assay using a polyclonal antibody against pneumococci was used to test formalin-fixed, paraffin-embedded tissue samples from 46 patients for whom bacterial culture results were available. Samples from 26 patients demonstrated pneumococcal antigens in areas of pneumonia, meningitis, or osteomyelitis or within circulating inflammatory cells. Various specimens from 18 patients grew S pneumoniae, whereas 8 had cultures that grew mixed bacteria or had no growth but were polymerase chain reaction-positive for the S pneumoniae Ity A gene. Pneumococcal antigens were not present in 20 cases (7 grew Streptococcus pyogenes; 9, Staphylococcus aureus; and 4, Haemophilus influenzae). Compared with culture, the immunohistochemical assay showed 100% sensitivity and 71% specificity. Immunohistochemical analysis has the diagnostic advantage of correlating host inflammatory reaction with presence of pneumococci.