Aspiration of the exudate through open perforation was performed in 54 children with chronic suppurative otitis media. Eighty aerobic and 81 anaerobic isolates were recovered. Aerobic bacteria only were involved in 20 patients (37%), and anaerobic organisms only in seven (13%). Mixed aerobic and anaerobic isolates were recovered from 27 patients (50%). The most common bacteria isolated were anaerobic gram-positive cocci, Bacteroides melaninogenicus group, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus. There were 45 beta-lactamase-producing bacteria (BLPB), 30 aerobes, and 15 anaerobes recovered from 38 patients (70%). beta-Lactamase activity was detected in 30 of the 38 ear aspirates (79%) that contained BLPB. All but one of these organisms were in excess of 10(4) colony-forming units/mL. The detection of beta-lactamase activity in the ear aspirates provides evidence of the role of BLPB in the failure of penicillin therapy to eradicate chronic ear infection.