Bacteriologic investigations were performed on 100 children with acute otitis media by culturing the fluid from a myringotomy site. Patients ranged in age from 7 months to 14 years, and 91 were younger than 6 years old. Bacterial isolates were yielded from cultures in 83 cases, and mixtures of two or three organisms were obtained from 15 patients. Among the total of 100 isolates, the most predominant organism was Streptococcus pneumoniae (28), followed by Hemophilus influenzae (26), Staphylococcus aureus (19), and Streptococcus pyogenes (six). Minimal inhibitory concentrations (MICs) of ampicillin sodium against S pneumoniae and H influenzae were 0.016 to 0.032 and 0.25 to 0.5 mg/L, respectively. None of the strains of H influenzae were resistant to ampicillin. The MICs of dicloxacillin sodium, cephalexin monohydrate, cefaclor, and erythromycin to H influenzae were 8 to 32, 8 to 16, 2 to 8, and 2 to 8 mg/L, respectively. The preferred drug for acute otitis media would be ampicillin in Japan, but we have to consider antistaphylococcal antibiotics for the patients who do not respond to 48 hours of treatment.