Objective: To identify the pathogens isolated from children with acute otitis media who did not respond to antimicrobial drug therapy. Methods: Retrospective analysis of cultures obtained by tympanocentesis from 46 children. Results: Organisms were recovered from 34 children (74%), and 43 isolates were recovered from these individuals. The organisms were Streptococcus pneumoniae (16 isolates), Haemophilus influenzae non-type b (12 isolates), Moraxella catarrhalis (5 isolates), Streptococcus pyogenes (5 isolates), Staphylococcus aureus (3 isolates), and Peptostreptococcus species (2 isolates). Resistance to the antimicrobial agent used was found in 27 (63%) of 43 isolates found in 22 patients (48%). Of patients who did not respond to amoxicillin therapy, H influenzae predominated. Streptococcus pneumoniae was recovered from 5 (56%) of 9 of those who did not respond to trimethoprim and sulfamethoxazole therapy, 4 (44%) of 9 patients after azithromycin therapy, 3 (25%) of 12 patients after amoxicillin therapy, and 2 (40%) of 5 patients after cefixime therapy. Streptococcus pyogenes was recovered from 2 (40%) of 5 patients after trimethoprim and sulfamethoxazole therapy and from 2 (40%) of 5 patients after cefixime therapy. Conclusions: The data illustrate the relation between resistance to antimicrobial drug therapy and failure of patients with otitis media to improve. They also highlight the importance of diagnostic tympanocentesis in establishing the presence of resistant microorganisms.