Chlamydia trachomatis is known to be a cause of acute non-gonococcal urethritis (NGU), though the aetiology of this disorder is not fully understood. Mycoplasma genitalium has been isolated from a few men with NGU, but culture has remained difficult and reliable detection became possible only with a specific polymerase chain reaction (PCR). We have used the PCR to examine the role of M genitalium in NGU. M genitalium was detected in urethral samples from 24 (23%) of 103 men with symptoms, signs, or both, of acute NGU, but from only 3 (6%) of 53 men without NGU (p < 0.006). This association was independent of the presence of C trachomatis and could not be explained by differences in age, ethnic, origin, lifetime number of sexual partners or a change in sexual partner during the previous 3 months. The clinical response of the mycoplasma-positive men to doxycycline treatment was at least as satisfactory as that of the chlamydia-positive men. These findings suggest that the association of M genitalium with NGU is likely to be causal, a notion consistent with the known virulence characteristics of this microorganism and its ability to cause urethritis in male sub-human primates.