BACKGROUND--Male urethritis may be caused by mycoplasmas. Since Mycoplasma genitalium has previously been isolated from the urethra of two men with non-gonococcal urethritis (NGU), it was the aim of the study further to elucidate its role by measuring the prevalence of this organism in men with NGU. MATERIAL AND METHODS--The polymerase chain reaction was used. Two different sequences of the gene coding for the main adhesin MgPa were amplified. Urethral, rectal, and throat samples from 99 male sexually transmitted disease (STD) patients with and without urethritis were studied. RESULTS--M genitalium DNA was demonstrated in 17/99 (17%) of the urethral swabs, but in none of the rectal and throat swabs. Significantly more patients with urethritis (13/52) were positive for M genitalium DNA than were patients without urethritis (4/47) (p < 0.03). In those with urethritis M genitalium DNA was found more often in Chlamydia trachomatis negative NGU (12/34) than in those with chlamydial NGU (1/14) (p = 0.05). Attempts to culture M genitalium from the PCR positive specimens were unsuccessful. CONCLUSION--M genitalium DNA was found significantly more often in male STD patients with non-chlamydial NGU than in men with chlamydial urethritis (p = 0.05) and in men without urethritis (p = 0.003), suggesting that M genitalium may be a cause of NGU. M genitalium DNA was not demonstrated in any of the throat or rectal swabsindicating that the urogenital tract is probably the primary site of infection or colonisation of this species.