We have previously reported Chlamydia pneumoniae (Cp) as a common pathogen in community-acquired pneumonia in children as young as 3 to 5 years old. We evaluated the middle ear fluid (MEF) from 35 children with acute otitis media for evidence of Cp. These MEF samples were cultured for Cp in cycloheximide-treated HEp-2 cells and were also evaluated with polymerase chain reaction (PCR) for Cp. Cp was isolated by culture in 5 MEF from 3 (9%) of 35 children, ages 8, 46 and 55 months, respectively. Cp was isolated from both ears in 2 children, who were also PCR positive. In the 3rd child, Cp was isolated from only one ear, which was PCR negative. Cp was a co-pathogen along with highly penicillin-resistant pneumococcus and penicillin-susceptible pneumococcus in 2 children; and the sole pathogen in 1 child. Lower respiratory diseases was not evident in any of the 3 Cp positive children. The other 32 children, whose ages ranged from 5 to 167 months, were both culture and PCR negative for Cp. The causative bacterial pathogens in the Cp culture negative children included: penicillin-susceptible pneumococcus (15), penicillin-resistant pneumococcus (8), Hemophilus influenzae (9),Moraxella catarrhalis (5), and Group A streptococcus (2). Seven patients had 2 or more organisms; only 2 had lower respiratory tract symptoms. This preliminary study is the first to report Cp as a potential pathogen of acute otitis media and suggests that this pathogen may be more common in pre-school children than is previously believed. Because Cp is susceptible only to macrolide/azolide antibiotics for children, the role of Cp in acute otitis media merits further evaluation. This study was supported in part by grants from Abbott Laboratories and Pfizer.