['Pathogen eradication in patients with acute otitis media (AOM) is associated with a reduced risk of clinical failures, but most children in whom middle ear fluid (MEF) culture remains positive show clinical improvement or clinical cure. We investigated the relationship between MEF culture-positivity during treatment in patients with clinical improvement/cure, and the occurrence of subsequent AOM.', 'A total of 673 patients with culture-positive MEF were enrolled in double-tympanocentesis studies and followed for 3 weeks after completion of treatment.', 'On day 4-6, 189/673 (28%) patients had culture-positive MEFs. Patients with clinical improvement/cure on day 11-14 (end of treatment) despite having culture-positive MEF on day 4-6 more often had recurrent AOM episodes (53/151, 35%) than those with culture-negative MEF (114/476, 24%; P = 0.007). 41/53 (77%) culture-positive patients with clinical improvement/cure on day 11-14 underwent tympanocentesis when AOM recurred and 29/41 (71%) were culture-positive. Pulsed field gel electrophoresis identity between pathogens at recurrence and those persisting on day 4-6 was found in 19/29 (66%) compared with 31/86 (36%) of the evaluable patients with recurrence and culture-negative MEF on day 4-6 (P = 0.005).'] Is persistence of pathogens despite clinical improvement in antibiotic-treated acute otitis media associated with clinical and bacteriologic relapse?