During a 13-month period ending in January, 1995, we obtained 159 samples of middle ear exudate through tympanocentesis (n = 155) or acute spontaneous otorrhea (n = 4) from 151 children enrolled in therapeutic trials of acute otitis media in a pediatric practice in Northern Virginia. Their ages ranged from < 1 to > 6 years of age (mean, 35 months; median, 22 months). Precise diagnostic criteria for acute otitis media always included bulging outward of all or part of the eardrum, opacification of the eardrum regardless of color and impaired mobility to positive and negative pressure via the pneumatic otoscope. Bacterial pathogens were isolated from middle ear fluid in 95% of these children: Streptococcus pneumoniae was recovered from 61 (37%); Haemophilus influenzae from 45 (27%); Moraxella catarrhalis from 41 (25%); Group A streptococcus from 6 (4%); Staphylococcus aureus from 4 (2%); and no growth or microbes of uncertain significance from 8 (5%). Six of the patients had mixed bacterial cultures; 2 of the 6 had at least one ampicillin-resistant bacteria, and a third had 2 ampicillin-resistant bacteria. Eight patients who failed to improve with antimicrobial treatment had a second tympanocentesis performed or developed spontaneous drainage; on that follow-up culture 3 of 8 cultures had different microorganisms; and 5 of the 8 bacterial specimens were resistant to ampicillin or penicillin. Twenty-one percent of the S. pneumoniae strains recovered from the middle ear were resistant to penicillin. Sixty-two percent of the H. influenzae and 98% of the M. catarrhalis isolates were resistant to ampicillin. Overall bacteria resistant to penicillin or ampicillin were recovered in 54% of middle ear fluid from 46 patients who had received a beta-lactam antibiotic in the preceding month as well as in 57% of middle ear fluids from 105 patients who had not. The empiric use of amoxicillin for treatment of acute otitis media should be reexamined in our community particularly in those who appear ill, have a high fever or have severe unremitting otalgia.