A polymerase chain reaction (PCR) assay based on the amplification of pneumolysin gene fragments in patient sera was developed for the etiologic diagnosis of acute pneumococcal pneumonia. Two pairs of oligonucleotide primers were designed to amplify a 348-bp and a 208-bp fragment of the pneumolysin gene. Amplified products were analyzed by agarose gel electrophoresis and hybridization using a 24-mer probe internal to the amplified DNA. The nested PCR could detect 10 organisms as determined by serial dilutions of DNA from Streptococcus pneumoniae. All 20 serum samples from patients with acute pneumococcal pneumonia (confirmed by blood culture) were positive. Among 100 serum samples from healthy elderly controls, 94 were negative and 6 were positive by PCR. Thus, PCR may be a novel diagnostic method for pneumococcal pneumonia.