We studied Mycoplasma pneumoniae as the etiologic pathogen in acute exacerbations of asthma and the value of polymerase chain reaction (PCR), culture, and serologic tests for its accurate diagnosis. For the study, 59 nonsmoking patients with asthma (37 females, 22 males; age, 15-50 years) underwent clinical, radiologic, and laboratory examinations. Bacteria isolated from sputum were Streptococcus pneumoniae (32 [54%]), Staphylococcus aureus (23 [39%]), and M pneumoniae (5 [15%]). All M pneumoniae were associated with S pneumoniae (8/32 [25%]) and S aureus (1/23 [4%]). No M pneumoniae were isolated as single pathogens. Serologic testing for M pneumoniae revealed that all samples were positive for specific IgG; 40 (68%) had a high titer, and 19 (32%) had a moderate titer. Of 59 samples, 29 (49%) were positive by Serodia Myco II gelatin particle testing (Fujirebio, Tokyo, Japan). PCR was positive in 25 samples, all of which had a high IgG titer; all culture-positive cases were PCR+. M pneumoniae is a common bacterial pathogen associated with acute exacerbations of asthma in people 15 years or older. Prompt laboratory diagnosis of M pneumoniae requires direct detection by PCR and culture. A high serologic titer can be a clue for the presence of M pneumoniae.