Neonatal otitis media may be an isolated infection or part of a more complex sepsis syndrome. Because early studies suggested that the microbiologic characteristics of neonatal otitis media included a high risk of gram-negative coliforms and Staphylococcus aureus, tympanocentesis was recommended routinely. Subsequent studies have supported empiric medical therapy in selected patients, reserving tympanocentesis for patients in whom medical treatment fails. Because of these conflicting recommendations, records from 37 neonates with otitis media who underwent tympanocentesis from 1986 through 1991 were studied retrospectively. Cultures in one outpatient (11%) and four inpatients (13%) yielded Escherichia coli, all of which were sensitive to amoxicillin. No cultures of S aureus were identified. Sterile cultures and pneumococcus isolates were found most frequently. No septic or intracranial complications were noted. All patients were discharged on regimens of routine antibiotics for otitis media.