Urethral swab specimens obtained from 101 men attending an STD clinic were examined for the presence of Mycoplasma genitalium by polymerase chain reaction (PCR) amplification. Fifty patients had non-gonococcal urethritis (NGU), and 51 patients were included as controls without urethritis. M. genitalium DNA was detected in 13 (26%) of the urethritis patients and in 5 (10%) of the control patients (P=0.06). No patient positive for M. genitalium had a simultaneous chlamydial infection. Thus, in the 36 patients with non-chlamydial NGU, the prevalence of M. genitalium infection was 36% (P=0.007 compared with controls). All patients with M. genitalium positive urethritis had a high grade urethritis defined as >10 polymorphonuclear cells per high power microscopical field. Compared with the control group, those with M. genitalium positive urethritis had more often had a history of urethritis than had those with chlamydial NGU or those with M. genitalium negative, non-chlamydial NGU.