Streptococcus pneumoniae is one of the important human pathogens in clinical microbiology. A polymerase chain reaction assay was designed to detect and identify S. pneumoniae through amplification of the ribosomal DNA spacer regions between the pneumococcal 16S-23S ribosomal RNA genes. Thirty-two Streptococcus and non-Streptococcus strains were tested to verify the specificity of the assay, and only S. pneumoniae strains gave a positive reaction. This method is a powerful technique for the rapid identification of S. pneumoniae.