The products of polymerase chain reaction (PCR) (1) are usually visualized by agarose or polyacrylamide gel electrophoresis followed by ethidium bromide staining. However, depending on the purpose of experiment, it sometimes needs to be shown whether the amplified DNA originated from target region or not. In that case a labelled oligonucleotide probe complementary to an appropriate internal sequence between the two normal primers is prepared and used as a probe for Southern hybridization (2). Another method uses a second PCR with another primer in the target sequence (3). The product is verified by restriction endonuclease digestion. However, confirmation by these methods is time-consuming, especially the Southern hybridization analysis. A new method of PCR using three primers is demonstrated in the present report. Amplification of DNA was performed based on the method described by Saiki et al. (1). Template DNAs were isolated from Mycoplasma hominis, M.pneumoniae, and Escherichia coli by the method of Marmur (4). Primers were obtained from 3 regions in 16S ribosomal RNA (rRNA) sequence of Mycoplasma species (Fig. 1). Two primers, B-1 and Mp-7, were demonstrated to be well-conserved regions among many bacterial 16S rRNAs (5), and used as outer primers in the present study. An inside primer, Mp-6, was shown to be specific for M.pneumoniae. A 50 Al of reaction buffer (10 mM Tris-Cl, 50 mM KCI, 1.5 mM MgCl2, 0.01% gelatin, pH 8.3) containing 200 ,iM dNTPs, 100 ng of template DNA, 2.5 units Taq polymerase (Stratagene, California, USA) and 1 AM outer primers