Objective: To investigate the effect of time since last urination on chlamydial positivity rates in men. Methods: Prospective study on men attending a genitourinary medicine clinic who were asked for the last time of their urination before obtaining their urine sample for testing for Chlamydia trachomatis with the Cobas Amplicor polymerase chain reaction (PCR) assay. Results: Of the total of 1649 men studied, 621 (37.6% (95% CI 35.3% to 39.9%)) had a voiding interval of less than 2 hours. There was no statistical evidence of a difference in the positivity rate of chlamydial infection among men with a voiding interval of less than 2 hours (106/621, 17.1%) and that of men with a voiding interval of 2 hours or longer (170/1028, 16.5%); difference in proportions 0.5% (95% CI to 3.2% to 4.3%), p = 0.779. Conclusion: Voiding interval does not have a significant impact on the performance of the Cobas Amplicor PCR assay in men.