BACKGROUND Chlamydia trachomatis is responsible for 30 to 50% of genital tract infections and is present, without symptoms, in 20% of men and 60% of women. We have little information in Chile about the prevalence of this infection. OBJECTIVE To assess the prevalence of Chlamydia trachomatis in men, using first catch urine samples. METHODS Three hundred and fifty one first catch urine samples of asymptomatic men and 50 samples coming from men with a primary urethritis, were analyzed. Urethral discharge samples from the latter were simultaneously studied. Analysis was performed using an enzyme immuno analysis (MicroTrak Chlamydia EIA, Syva Co.) and a nested polymerase chain reaction towards the gene that codifies MOMP (PCR/OMP). RESULTS Among asymptomatic men, two of 154 teenagers aged 18 to 19 years old (1.3%), 10 of 100 university students (10%) and eight of 97 adults over 30 years old (8.2%), were infected. The global prevalence of infection in these men was 5.7%. The prevalence of infection in men with urethritis was 12%. Urine EIA had a higher detection frequency than PCR/OMP, but according to another PCR assay, these results were false positives. EIA in first catch urine, had a sensitivity, specificity, positive and negative predictive value of 83.3, 75, 31.3 and 97% respectively, for the detection of Chlamydia trachomatis. The figures for PCR/OMP were 100% for all these parameters. CONCLUSIONS The prevalence of Chlamydia trachomatis infection in Chilean men is similar to that reported in developed countries. Enzyme immuno assay in first catch urine had a good diagnostic accuracy and could be used in epidemiological studies in asymptomatic men.