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Trimethoprim sulfamethoxasole has been recommended for the treatment of acute otitis media. In this double tympanocentesis study, children aged 3 to 48 months with acute otitis media received trimethoprim sulfamethoxasole twice daily (40 mg/kg/d) for 10 days. All children had a baseline tympanocentesis and in culture-positive children, tympanocentesis was repeated at the on-therapy visit. Of 89 children enrolled, 51 (57%) were clinically and bacteriologically evaluable. Bacteriologic eradication was achieved in 80% (42 of 52) of children, and overall clinical response at the end of therapy was 78%. Clinical success was 69% for culture-positive children versus 91% for culture-negative children at baseline tympanocentesis (P = 0.03). In this study, trimethoprim sulfamethoxasole clinical response was unsatisfactory, especially among culture-′positive children.