- Background. Because of the increasing number of resistant middle ear pathogens and the impact of the new conjugate Streptococcus pneumoniae vaccine, an active surveillance of the microbiology and susceptibility pattern of middle ear pathogens is required. Objective. To study the microbiology and susceptibility pattern of middle ear pathogens obtained from Costa Rican children with acute otitis media (AOM), recurrent otitis media (ROM) and therapeutic failure otitis media (FOM). Methods. Between 1999 and 2001 middle ear fluid (MEF) was collected from 276 Costa Rican patients. S. pneumoniae serotyping and pulsed field gel electrophoresis analysis was done on available strains. Results. Among the total study population, 102 were AOM patients, 98 were ROM patients and 76 were FOM patients. Overall S. pneumoniae (88 strains) was the most common pathogen isolated followed by Haemophilus influenzae (41 strains) and Streptococcus pyogenes (10 strains) . H. influenzae was the most common agent in FOM patients (P = 0.015). Beta-lactamase production was observed in 3 of 41 (7%) H. influenzae strains and 3 of 3 (100%) Moraxella catarrhalis strains. Penicillin-nonsusceptible S. pneumoniae strains were more common in FOM (64%) and ROM (63%) patients than in AOM (42%) patients (P = 0.05). S. pneumoniae serotype 19F was the most prevalent serotype, mainly within one distinct clone. Conclusions. Overall S. pneumoniae serotype 19F was the most common isolate from the middle ear fluid of Costa Rican children. Beta-lactamase-negative H. influenzae was the most prevalent in the subpopulation of patients with FOM. S. pyogenes was the third most common isolate and M. catarrhalis was uncommon.