Mycoplasma genitalium was first isolated from two men with non-gonococcal urethritis (NGU) and thereafter shown to produce urethritis in subhuman primates, inoculated intraurethrally. This mycoplasma has been detected significantly more often in patients with acute NGU, particularly in patients with non-chlamydial NGU, than in subjects without urethritis. The prevalence of M. genitalium-positive non-chlamydial NGU ranges from 18 to 46% of all non-chlamydial NGU cases. In addition, the persistence of M. genitalium in the urethra after antimicrobial chemotherapy is associated with persistence or recurrence of NGU. The various results reported to date tend to support the proposition that M. genitalium is a pathogen of NGU. M. genitalium is highly susceptible to tetracyclines, macrolides, and some new fluoroquinolones, but the clinical data on the chemotherapy in M. genitalium-positive NGU is extremely limited. Because of the possible association between the post-treatment presence of M. genitalium in the urethra and persistent or recurrent NGU, the eradication of this mycoplasma from the urethra is essential in the management of patients with M. genitalium-positive NGU. Further studies are required to establish the optimal chemotherapy for M. genitalium-positive NGU.