The aim of this retrospective study was to determine the possible causes of failure of antibiotic therapy in children with acute otitis media (AOM). Thirty-nine samples of middle-ear fluid were obtained by myringotomy from 31 children suffering from AOM, unrelieved by antibiotic therapy administered for over 48 hours. The samples were analysed by the usual microbiological techniques, including cultures, tests for beta-lactamase producing strains and the determination of the minimal inhibitory concentration of penicillin for Streptococcus pneumoniae. In 14 samples, no bacterial strains were detected in the cultures of middle-ear fluid; and in two samples the cultures revealed two strains of bacteria. The bacteria most frequently identified were Haemophilus influenzae, found in 11 samples, and Streptococcus pneumoniae, found in seven samples, of which four produced strains with reduced susceptibility to penicillin. The failure of antibiotic therapy in AOM appears to be related to the increased resistance of Haemophilus influenzae and to the reduced susceptibility of Streptococcus pneumoniae to penicillin. Other factors contributing to the failure of antibiotic therapy in AOM may be the viruses or the bacteria that produce multiple pathogens in the middle ear.