Real-time PCR was employed to detect a region of the Mycoplasma genitalium mg219 gene, a gene of unknown function, in clinical samples. Amplification of DNA and signal production from 15 other species of human mycoplasmas and 14 other bacteria and viruses did not occur. Using a panel of 208 genital and rectal samples, the sensitivity when compared to the modified mgpa gene (encoding the major surface protein MgPa) real-time PCR assay was found to be 100% and the specificity of the assay 99.5% with a positive predictive value of 80% and a negative predictive value of 100%. The mg219 gene was found to be in all strains of M. genitalium and was highly conserved. M. genitalium was detected in 3.9% (11/280, 95% CI 2.1-6.9) of all male specimens, in 7.7% (10/130, 95% CI 4.1-13.7) of patients with non-gonococcal urethritis (NGU) and in 0.7% (1/150, 95% CI <0.01-4.1) of patients without urethritis. The presence of M. genitalium was significantly associated with NGU (P < or =0.01; 95% Cl 0.88-0.98) and non-chlamydial-non-gonococcal urethritis (P=0.0005; 95% Cl 0.84-0.97).