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Objective: To determine the association of Mycoplasma genitalium and Ureaplasma urealyticum in symptomatic male patients presenting with non-gonococcal urethritis in a sexually transmitted infection clinic in Hong Kong., Design: Cross-sectional study., Setting: A sexually transmitted infection clinic, Department of Health, Centre for Health Protection, Hong Kong., Patients: A cohort of consecutive new male patients attending the government sexually transmitted infection clinic., Main Outcome Measures: Prevalence of Mycoplasma genitalium and Ureaplasma urealyticum among symptomatic male patients with non-gonococcal urethritis and asymptomatic patients without non-gonococcal urethritis., Results: Specimens of 22 and 10 patients tested positive by polymerase chain reaction for Ureaplasma urealyticum and Mycoplasma genitalium respectively, among the symptomatic non-gonococcal urethritis group (n=98). In the asymptomatic control group (n=236), corresponding patient numbers whose specimens tested positive were 47 and 5. There was no statistically significant difference between the two groups, in terms of the proportion of patients infected with Mycoplasma genitalium (P=0.799) or Ureaplasma urealyticum (P=0.535)., Conclusions: In our study, demonstration of Mycoplasma genitalium and Ureaplasma urealyticum by polymerase chain reaction was not associated with symptomatic non-gonococcal urethritis in male patients attending a Hong Kong government clinic for sexually transmitted infections.