PURPOSE: Pleural effusions may develop during the course of bacterial pneumonia. The aim of this study was to evaluate the significance of the polymerase chain reaction (PCR) method for detection of Mycoplasma pneumoniae, Mycobaterium tuberculosis and Staphylococcus aureus from pleural fluid. METHODS: Total 12 samples were obtained from pleural fluid; 2 samples from children with Mycoplasma pneumonia, 5 samples from adults with tuberculous pleurisy, and 5 samples from sterile pleural fluid seeded artificially with staphylococcus aureus. The primers used for our PCR were prepared to amplify M. pneumonia-specific MP5 gene, M. tuberculosis-specific IS6110 gene, and S. aurus-specific femA and mecA gene. The amplified PCR products were detected by ethidium bromide-stained agarose gel electrophoresis. RESULTS: A total of 12 pleural fluid samples were tested by nested PCR using the specific primer set. We could amplify MP5 gene in 2 samples, IS6110 gene in 5 samples, mecA gene in 3 samples, and femA gene in 5 samples. These PCR data were correlated with serolological data, microbiological data and methicillin-sensitivity test result. There were no false-positive results due to cross-contaminating DNA between these 3 organisms. CONCLUSIONS: We conclude that enzymatic amplification of specific gene from pleural fluid might be useful to diagnose the infectious pleural effusion by Mycoplasma pneumoniae, Mycobacterum tuberculosis or Staphylococcus aureus.