Mycoplasma pneumoniae causes bronchitis and pneumonia predominantly in subjects 5 to 20 years old. M. pneumoniae is detected by measuring specific antibodies and/or isolating the microorganism, but the frequency of false-positive/negative results, and the culture time required until isolation pose problems. We detected M. pneumoniae using real-time PCR with clinical specimens. We also determined the drug sensitivity of isolated M. pneumoniae and searched for the gene mutation responsible for macrolide resistance. In 275 cases of suspected M. pneumoniae infection, positive cases in real-time PCR numbered 40 (14.5%). Of these, 16 showed positive culture (5.8%). Of these 16, A2063G point mutation that causes macrolide resistance was found in 12. Drug sensitivity testing showed resistance to clarithromycin (MIC> or =64 microg/ml) in 11 and susceptibility in 4 (MIC 0.0039 microg/ml). The clarithromycin resistance ratio was 75%. Growth was insufficient for testing in 1 case. M. pneumoniae was susceptible to minocycline and all quinolone drugs. M. pneumoniae detection using real-time PCR proved much more sensitive than conventional culture. Macrolide resistance results correlated well with genomic mutation. Our study's macrolide resistance ratio was high at 75% possibly due to a restricted subject population that had been administered macrolide drugs elsewhere but with an unsatisfactory outcome. The increasing number of reports on macrolide resistance requires that we monitor drug resistance trends, particularly among macrolide derivatives.