Urogenital infection with Chlamydia trachomatis in adults and adolescents is a common sexually transmitted disease. The purpose of this study was to investigate whether isolated microhematuria in children and adolescents is associated with Chlamydia trachomatis infection of the urinary tract. The study group included 37 children and adolescents with isolated nonglomerular microhematuria. Urethral smears for the isolation of Chlamydia trachomatis in cell culture were taken at the time of cystourethroscopy from all patients. Polymerase chain reaction (PCR) for the detection of chlamydial DNA in urine was carried out in 25 of 37 (68%) patients and direct immunofluorescence (DIF) of urine in 16 of 37 (43%) patients. The control group included 33 children and adolescents without hematuria; PCR and DIF of urine were carried out in all controls. Chlamydia trachomatis infection of the urinary tract was confirmed in 8 of 37 (22%) patients in the study group, and in none in the control group (0 of 33, P<0.001). Further studies of larger groups of patients should be conducted, before recommending testing for Chlamydia trachomatis infection of the urinary tract in children and adolescents with unexplained microhematuria.