The aetiology of urethritis, the significance of potential pathogens and the relation of urethritis to HIV infection were determined in 335 men (cases) with and 100 men (controls) without urethral symptoms. Urethral swab specimens were tested for different organisms by PCR or by culture for Neisseria gonorrhoeae. The prevalence of N. gonorrhoeae and Chlamydia trachomatis was 52 and 16%, respectively. The potential pathogens: Mycoplasma genitalium, Ureaplasma urealyticum, Trichomonas vaginalis and herpes simplex virus (HSV), were present in 5, 36, 6 and 6% of the cases respectively. M. genitalium was the only potential pathogen associated with microscopic urethritis. After excluding gonococcal infections, U. urealyticum was more frequent in symptomatic patients, while the prevalence of T. vaginalis was similar among cases and controls. These results strongly suggest an a etiological role for M. genitalium in male urethritis, a possible role for U. urealyticum, but not for T. vaginalis. The control group, with 97% genital ulcer disease patients, was not suitable for the investigation of the role of HSV. The sero-prevalence of HIV was 45%. Current infections were not associated with HIV. However, a history of previous urethral discharge was associated with HIV in a multivariate analysis and supported the hypothesis that non-ulcerative sexually transmitted diseases facilitate HIV transmission.