In this prospective study tympanocentesis was performed to determine the pathogens isolated from middle ear fluid of 200 ears in 137 children with acute otitis media (AOM) which had not responded after one or two empiric antimicrobial treatment courses (termed persistent AOM). For comparison tympanocentesis from 154 ears in 111 children with AOM not previously treated are described. Patients were enrolled from October, 1989, until September, 1992. In the persistent AOM group amoxicillin and trimethoprim/sulfamethoxazole were the most frequently used antimicrobials before tympanocentesis. Middle ear aspirates produced no pathogenic bacterial growth in 49% of persistent AOM patients, Streptococcus pneumoniae in 24%, Haemophilus influenzae in 7%, Branhamella catarrhalis in 7%, Streptococcus pyogenes in 6%, Staphylococcus aureus in 5% and two pathogens in 3%. Two (18%) of 11 S. pneumoniae isolates tested were penicillin-resistant; 1 was intermediate and 1 was highly resistant. Ten (83%) of 12 H. influenzae and all of 11 B. catarrhalis AOM isolates produced beta-lactamase. In comparison previously untreated AOM patients produced no bacterial growth from tympanocentesis in 30%, S. pneumoniae in 36% (8% penicillin-resistant), H. influenaze in 13% (44% beta-lactamase-producing) and B. catarrhalis in 11% (85% beta-lactamase producing). AOM which is persistent after initial empiric antimicrobial therapy may be caused by middle ear inflammation after bacteria are killed or involve penicillin-resistant S. pneumoniae, beta-lactamase-producing H. influenzae or B. catarrhalis more commonly than occurs in AOM which has not been recently treated.