For many years, trimethoprim-sulfamethoxazole (TMP-SMX) has been recommended as an alternative antimicrobial agent for the treatment of children with otitis media (OM). This study analyzed the in vitro activity of TMP-SMX against respiratory pathogens obtained from middle ear fluid of Costa Rican children 6-60 months of age with acute OM, recurrent OM, therapeutic failures and acute OM at risk for having a resistant pathogen. Between 2002 and 2003, a total of 124 middle ear fluid bacterial isolates were analyzed and compared with historic data from 1992 to 1997. A significant increase in the number of TMP-SMX Streptococcus pneumoniae (P = 0.00008)- and Haemophilus influenzae (P = 0.04)-resistant strains was observed during 2002-2003 when compared with strains from 1992-1997.