Male urethritis is one of the most common sexually transmitted infections (STIs). However, the aetiology is still unclear in many cases. In this study the prevalences of Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV-1), HSV-2, cytomegalovirus (CMV), adenovirus, Chlamydia trachomatis, Mycoplasma genitalium and Ureaplasma urealyticum (including subtyping) were investigated. Samples from 112 male STI attendants with microscopically verified urethritis and from a control group of 103 men without clinical or microscopic signs of urethritis were analysed. Prevalences in the urethritis group compared with the controls were as follows: EBV 21%, 6% (P < 0.01); C. trachomatis 15%, 3% (P < 0.01); M. genitalium 6%, 1% (P = 0.067) and U. urealyticum 10%, 10% (ns). The results for HSV-1, HSV-2, CMV and adenovirus were negative in patients, and therefore not analysed in the controls. EBV was shown to be an independent predictor of urethritis and may play a role in its pathogenesis.