To test the ability of the polymerase chain reaction (PCR) to detect Streptococcus pneumoniae in blood, we generated two sets of nested primers. The first defined 559-bp and 649-bp regions of the pneumolysin gene, and the second defined 445-bp and 553-bp regions of the autolysin gene. These nucleotide segments were detected in DNAs from isolates of all 20 pneumococcal serotypes tested, but they were not detected when used to test DNAs from 41 isolates of nonpneumococcal bacteria and fungi. The sensitivity was evaluated by using purified pneumococcal DNA. We were able to detect 10 fg of S. pneumoniae DNA, or 4.3 genome equivalents. Blood samples were obtained from 16 patients with culture-proven pneumococcal bacteremia and were subjected to PCR analysis. Of eight buffy coat fractions tested, six showed reactivity in the PCR with the pneumolysin primers, and five of the eight produced the expected products when tested with the autolysin primers (sensitivities, 75 and 63%, respectively). Of the eight whole-blood specimens tested, only three produced the expected products with either set of primers. Additionally, we tested 14 samples from patients with bacteremia that were culture positive for nonpneumococcal bacterial species, and 13 were negative (specificity, 93%). This combination of sensitivity and specificity may make detection of S. pneumoniae in blood by PCR in comparison with that by blood culture a very promising alternative for a means of definitive diagnosis.