A 16S rDNA-PCR assay for Mycoplasma pneumoniae applied to nasopharyngeal secretion (NPS) or pharyngeal swab (PS) from children with community-acquired pneumonia (CAP) was prospectively compared to serological tests including complement fixation (CF) test, a mu-capture enzyme immuno assay (EIA) for the detection of specific IgM, and an EIA for the detection of specific IgG. During a 24-months-period diagnosis of active M. pneumoniae infection was established in 32 (12.6%) of 253 patients for whom paired sera were available. In the acute phase, the sensitivities of PCR from NPS and PS, CF test, IgM EIA, and IgG EIA were 90.0%, 79.3%, 46.9%, 78.1%, and 59.4%, respectively. The corresponding specificities were 98.1%, 98.6%, 97.6%, 87.1%, and 72.4%, respectively. Thus, the 16S rDNA-PCR assay provides a highly sensitive and accurate tool for the rapid diagnosis of M. pneumoniae infection in children with CAP.