Bacterial culture of middle-ear fluid (MEF), the standard for etiologic diagnosis of acute otitis media (AOM), has revealed Streptococcus pneumoniae (Pnc) to be a major pathogen responsible for one-third of AOM cases. In the present study, we compared the results of polymerase chain reaction (PCR) based on the amplification of the pneumolysin gene with the results of pneumococcal culture, for 2595 MEF samples obtained during AOM events in 831 children who were followed from 2-24 months of age in the Finnish Otitis Media Vaccine Trial. PCR results were positive for 47.1% of the MEF samples, and culture results were positive for 27.3% of the samples. PCR-positive, culture-negative samples were associated with previous Pnc AOM in a time-dependent pattern, concurrent antibiotic treatment, low volume of MEF, and concurrent nasopharyngeal carriage. PCR-positive AOM represented a clinically less severe disease, compared with culture-positive Pnc AOM. A positive PCR result seemed to indicate the presence of viable, although often nonculturable, Pnc.