OBJECTIVE To further understand the roles of bacteria and antibiotics in the development of otitis media with effusion (OME). METHODS Samples of middle-ear effusion (MEE) were collected during the placement of ventilation tubes to treat chronic OME. Children with acute otitis media within the past three months were excluded from this study. We used polymerase chain reaction (PCR) to detect pathogens and to test the susceptibility of Streptococcus pneumoniae to penicillin. RESULTS Among MEE samples from 52 children, PCR detected bacterial DNA in 32 per cent (24/75) of them. S. pneumoniae was detected more frequently in middle ears that required ventilation tube insertion at least twice compared with those requiring ventilation tube insertion only once (5/15 versus 4/60; p = 0.013). Higher levels of S. pneumoniae were detected in MEE from children with, than without, a long history of antibiotic administration (7/10 versus 2/14; p = 0.0187). The pbp genes of all isolated S. pneumoniae contained mutations. CONCLUSIONS Long exposure to antibiotics might significantly influence the bacterial genome in MEE.