Despite decades of careful study, the etiologies of all cases of pelvic inflammatory disease (PID) and non-gonococcal urethritis (NGU) have yet to be described. Mycoplasma genitalium is a newly described organism which has been implicated as a cause of both PID and NGU. Because of fastidious growth requirements, prolonged incubation time and frequent overgrowth in clinical specimens by Mycoplasma hominis, non-culture methods need to be developed for its detection. We have cloned M. genitalium DNA by transfection into Escherichia coli using M13 as the vector. Using these segments as templates, we synthesized radiolabelled cDNAs that were tested for specific hybridization with M. genitalium, and clinically isolated genital mycoplasmas presumptively identified as M. hominis, and Ureaplasma urealyticum. A 256 base-pair segment was found to hybridize with M. genitalium with a sensitivity of 10(2) colour-changing units (CCUs). No cross-hybridization was observed with M. hominis, and cross-hybridization was observed only with large concentrations (greater than 10(6) CCUs) of U. urealyticum. Because of our choice of M13 as the vector, which contains the Lac Operon of E. coli, slight hybridization occurred with E. coli as well. This cDNA can be used against clinical specimens to determine the ecologic niche and spectrum of disease caused by M. genitalium.