We evaluated a kit for the rapid diagnosis of Mycoplasma pneumoniae infection and investigated the antimicrobial susceptibility of the isolates. A total of 194 otherwise healthy children, aged 0.3–14.9 years, were diagnosed as having pneumonia by chest X-ray findings between December 2003 and November 2004, and were admitted to Showa Hospital. Isolation of M. pneumoniae was attempted from a throat swab obtained on admission, and the complement fixation titer was measured in paired serum samples obtained at admission and at the convalescent stage. We also used a rapid diagnosis kit (ImmunoCard Mycoplasma) for the detection of specific immunoglobulin M antibody in paired sera. Pneumonia due to M. pneumoniae was defined by isolation of this microorganism, or by seroconversion, or a ≥4-fold increase in the antibody titer. Using each isolate, we determined the minimum inhibitory concentrations for five antimicrobial agents by the broth dilution method. M. pneumoniae pneumonia was diagnosed in 45 children (23.2%). The ImmunoCard had a sensitivity of 31.8% using admission serum and 88.6% using paired sera, while the specificity was 78.1% and 70.5%, respectively. M. pneumoniae was isolated from 14 of the 45 patients (31.1%). The 50%/90% minimum inhibitory concentration (µg/ml) of erythromycin, clarithromycin, azithromycin, minocycline, and levofloxacin was 0.006/0.012, ≤0.003/≤0.003, ≤0.003/≤0.003, 0.78/1.56, and 0.39/0.78, respectively. For a rapid diagnosis of M. pneumoniae pneumonia, the ImmunoCard was not effective. Macrolides showed superior in vitro antimicrobial activity against M. pneumoniae isolates causing pediatric pneumonia.