In order to improve the diagnosis of a Mycoplasma pneumoniae infection, we developed a polymerase chain reaction (PCR)-based assay. The gene encoding elongation factor Tu (tuf) was selected as the target sequence. Oligonucleotides derived from variable stretches of the tuf gene were able to prime the amplification of a 950-bp fragment exclusively when M. pneumoniae DNA was used as the template. The sensitivity of the assay was increased 10-fold when the amplification products were hybridized with an internal M. pneumoniae-specific oligonucleotide. The use of three to four genome copies for PCR was sufficient for obtaining a hybridization signal. In addition, we substituted radioactive filter hybridization with a microtiter plate assay. Via a biotin moiety of one PCR primer, the amplification products were immobilized on streptavidin-coated microtiter plates. Subsequent hybridization with a digoxigenin-labeled oligonucleotide resulted in the same sensitivity and specificity as those obtained by filter hybridization. Clinical application of the assay was performed on 102 throat swab specimens from patients with respiratory tract infections. Of 21 culture-positive samples, 19 were confirmed to be positive in the PCR-based assay (sensitivity, 90%). Furthermore, 14 of 19 seropositive but culture-negative samples gave a positive hybridization signal. Of 62 culture-negative and seronegative specimens, 60 gave a negative result in our assay (specificity, 97%). Of the 33 samples that were positive in our PCR-based assay, 5 samples initially gave false-negative results because of the presence of inhibitory substances in those specimens. Inhibition of Taq polymerase in these five cases was prevented by an additional step of phenol extraction and subsequent ethanol precipitation.