Primers specific to conserved and variable regions in the 16S rRNA sequence were selected from the partially sequenced 16S rRNA genes of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, and Staphylococcus epidermidis. The PCR assay was divided into two DNA amplifications. The first resulted in a general bacterial amplicon, and the second resulted in a species-specific amplicon. The high specificity of the PCR assay was documented after testing bacteria of 28 different species (133 strains). A total of 304 clinical cerebrospinal fluid samples, including 125 samples from patients with bacterial meningitis, were assayed to investigate the diagnostic sensitivity and specificity for bacterial meningitis. The assay showed high sensitivity (0.94) and specificity (0.96) with the clinical samples, although some false results were obtained, the reasons for which are discussed. With agarose gel electrophoresis for detection of the PCR products, the detection limit for meningococci in cerebrospinal fluid was 3 x 10(2) CFU/ml.