Tympanocentesis with aerobic and anaerobic cultivation of middle ear fluid was performed through one or both tympanic membranes of 186 children with acute otitis media. Aerobic bacteria alone, predominantly pneumococcus and Hemophilus influenzae, were isolated from 118 (63.4%) patients; and anaerobic organisms alone, most commonly Peptococcus, from 24 (12.9%). Twenty-six (14%) yielded mixtures of aerobes and anaerobes, and several had multiple aerobic or anaerobic agents. No bacterial growth was noted in 18 (9.7%) patients. The isolation of anaerobic bacteria alone, supported in some patients by Gram-stain observations of middle ear fluid, suggests that these bacteria, especially Peptococcus, may occasionally play a direct role in acute otitis media.