The microbiology of otitis media (OM) has been documented by cultures of middle ear fluid (MEF) obtained by needle aspiration. The results of studies of bacteriology of OM from Sweden, Finland and the United States are very similar: Streptococcus pneumoniae and Haemophilus influenzae are the most important pathogens: Gram-negative enteric bacilli are isolated from MEF of approximately equal to 20% of infants to six weeks of age but are rarely present in MEF of older children; group A beta hemolytic streptococcus and Staphylococcus aureus are infrequent causes of otitis. Preliminary results suggest that anaerobic bacteria are responsible for some episodes of OM. Although epidemiologic data suggest that virus infection is associated with OM, the results of ten studies indicate that these agents are infrequently isolated from MEF of children with OM. Viruses were isolated from 29 to 663 patients (4.4%). Respiratory syncytial virus and influenza virus were isolated most frequently. The results of seven studies of mycoplasma infection in 771 patients with OM included only one isolation of Mycoplasma pneumoniae from MEF. These results indicate that viruses and mycoplasmas are uncommonly found in MEF of patients with OM, but few studies have been attempted in recent years. Chlamydia trachomatosis is the etiologic agent of a mild but prolonged pneumonia in infants. OM may accompany the respiratory infection, and C. trachomatis has been isolated from MEF of some of these infants. Recent studies of asymptomatic children with persistent MEF indicate that bacterial pathogens are present in some of these fluids. Investigators in Columbus, Boston, and Pittsburgh obtained MEF for culture at the time of myringotomy or placement of tympanostomy tubes. Bacteria were isolated from MEF of 50% of these children; S. pneumoniae, H. influenzae, or group A streptococcus were isolated from 10% to 20% of cases. There were only minimal differences in the rates of isolation of bacteria from serous, mucoid, or purulent fluids. The significance of these results is uncertain, but they suggest that the persistent effusion may be a result of asymptomatic but prolonged infection or may be an immune response to a persisting antigen.