By using the primers designed on the bases of the sequences of the 16S rRNA genes of Mycoplasma pneumoniae and Mycoplasma genitalium, respectively, specific and sensitive in vitro DNA amplification assay system for the detection and discrimination of these two mycoplasmas was established.The detection limit of the assay was 100 cells for M. pneumoniae and 1,000 cells for M. genitalium. Neither other human mycoplasmas nor oral bacteria existing in human saliva showed any crossreactions with these primers.Mycoplasma pneumoniae is a specific pathogen for humans causing respiratory infections as well as a wide range of extrapulmonary complications (5). The diagnosis of M. pneumoniae infection has been based on the direct isolation of M. pneumoniae cells from patient's throat by the culture method or on the detection of specific antibodies against M. pneumoniae in the patient's serum. However, the cultivation of M. pneumoniae is time consuming and requires about 2 to 3 weeks to get results.Recently, DNA hybridization techniques or a novel in vitro DNA amplification procedure through the polymerase chain reaction (PCR) were developed. M. pneumoniae-specific PCR assays have been reported using the primers directed against P1 adhesin gene (8), a specific DNA sequence from M. pneumoniae genomic library (2), and 16S rRNA gene (8). Primers for M. genitalium-specific PCR were also developed by using sequences of adhesin gene (9, 12). However, some commercial kit of DNA hybridization for M. pneumoniae (4) and the primers directed against 16S rRNA gene of M. pneumoniae (8) showed the cross-reactions with M. genitalium (16) which has several common properties with M. pneumoniae (3, 10, 13). We report herein specific in vitro DNA amplification assay for the detection and discrimination of M. pneumoniae and M. genitalium by using primers designed on the bases of the 16S rRNA genes of the two mycoplasmas. In order to develop primers which discriminate between M. pneumoniae and M. genitalium, we applied the theory of the allele-specific polymerase chain reaction (19). The allele-specific PCR, first developed for the diagnosis of sickle cell anemia, allows direct detection of the point 21