We compared a newly developed pneumococcal polymerase chain reaction (PCR) for Streptococcus pneumoniae (Pnc) to bacterial culture in 123 middle ear effusion (MEE) samples of 123 children with otitis media with effusion (OME). For the pneumococcal PCR assay, DNA of MEE samples was purified by a QIAamp blood kit. The outer primers used amplified a 348 basepair region of the pneumolysin gene, and the inner a 208. Pnc was cultured in 14 (11%) and pneumolysin PCR was positive in 57 (46%) of the 123 MEE samples. All the culture positive samples were also PCR-positive. Both the samples with culturable Pnc and with positive pneumolysin PCR increased with shorter duration of OME and a greater number of acute otitis media during the preceding 6 months. In conclusion, pneumolysin PCR suggests pneumococcal involvement in MEE even in OMEs with no evidence of Pnc in culture, and thus offers a good diagnostic tool when a more accurate and sensitive pneumococcal diagnosis is needed.