Otitis media with effusion (OME) is one of the major causes of hearing loss in childhood. The pathogenesis still remains unclear, though it is closely related to acute otitis media with bacterial infections. It is known that Haemophilus influenzae is one of the most common bacteria isolated from middle ear effusions (MEEs). Recently, in vitro DNA amplification by polymerase chain reaction (PCR) is a new technology that has considerable implication for diagnosis of viral and bacterial infections because of its potentially precise specificity and sensitivity. In the present experiment polymerase chain reaction (PCR) was applied to the detection of DNA genome of H. influenzae contained in middle ear effusions. By Southern blot hybridization, two characteristic bands for H. influenzae DNA were detected at 273 b.p. and 550 b.p. position in 15 of 27 MEEs. However, no organism was cultured by conventional methods. Our results indicate the PCR technique is more specific and sensitive in detection of bacteria in middle ear effusion of OME, compared with conventional methods. It strongly suggests more involvement of the bacteria, especially H. influenzae, in OME onset.