Male patients are assessed for a sexually transmitted infection provided a considerable length of time has elapsed since last micturition. The current availability of highly sensitive nucleic acid amplification tests (NAATs) for Chlamydia trachomatis (CT) and Neisseria gonorrhoea such as APTIMA Combo2 (AC2) led us to investigate the impact of voiding interval on the positivity of urine tests for CT. Male patients attending a genitourinary medicine clinic at high clinical risk for CT infection and known CT positives returning for treatment were included. Two first-void urine (FVU) specimens were collected: the first sample in the standard manner and the second sample was collected 20 minutes later or as soon as possible thereafter. Fifty-two CT-positive males were included in the analysis. All of the second samples were also CT positive and none were in the equivocal range. Paired t-test analysis did not show a significant difference between relative light unit readings of the first and second urine samples (P = 0.127). Even in male patients who have recently passed urine, FVU tested by AC2 can still reliably detect CT. This provides us opportunity for more flexible and effective patient management.