Our objectives were to study the distribution of Chlamydia trachomatis and Mycoplasma genitalium in men with or without non-gonococcal urethritis (NGU) and their respective female partners. A case-control study was carried out to which men with or without NGU and their female partners were recruited. All study participants were tested for the presence of C. trachomatis and M. genitalium. An analysis firstly of the distribution of each of these microorganisms among men with or without urethritis and their respective female partners was carried out. Furthermore, we examined the association of each of these microorganisms and NGU when the other had been excluded. Chlamydia trachomatis was present in 14 (36%) of 39 men with NGU compared to none of 12 men without NGU (P=0.022). The prevalence rates for female partners of men with NGU were 10 (26%) of 39 compared to none of 12 partners of men without NGU (P=0.092). M. genitalium was detected in 12 (33%) of 36 men with NGU compared to 1 (9%) of men without NGU (not significant; P=0.147). The prevalence rates for female partners of men with NGU were 10 (32%) of 31 women compared to none of 7 partners of men without NGU (not significant; P=0.156). There was a greater concordance than discordance of carriage of each of the 2 microorganisms among the study couples and each tended to be carried independently of the other by men. Analysis of the association between the presence of C. trachomatis in men and NGU was significantly improved by the exclusion of men with M. genitalium (P=0.0058). Likewise, the association between the presence of M. genitalium in men and NGU was significantly improved by the exclusion of couples in whom either the man or woman was C. trachomatis-positive (P=0.049). The independent carriage of C. trachomatis and M. genitalium by men with NGU, coupled with the improved association between each pathogen and NGU by exclusion of the other provides support for the separate role of each in the aetiology of NGU.