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Objective: To evaluate the effectiveness of systematic investigation for Group B Streptococcus (GBS) in urine samples to detect colonization in pregnant women., Methods: This study included 1036 pregnant women whose urine samples were cultured in our laboratory during 2006. Any colony consistent with GBS was identified in urine or in rectovaginal samples submitted for screening of GBS colonization., Results: GBS was recovered in urine samples from 111 of the 1036 women (10.7%), and in 77 of them bacterial count was <10(4) colony forming units/mL. Screening for GBS in rectovaginal samples was performed in 841 of the 925 pregnant women who did not have GBS bacteriuria (10.1% positive results) and in 61 of the 111 with GBS bacteriuria (60.7% positive results; no significant differences were found when results were stratified by colony count). Estimated rectovaginal colonization was 15.4%, and colonization exclusively detected in urine was 4.2%. Only 30% of pregnant women with GBS bacteriuria, but negative antenatal screening cultures who were admitted to our hospital for delivery received intrapartum antibiotic prophylaxis., Conclusions: Systematic investigation of the presence of GBS in urine samples from pregnant women improves the detection of carriers who are candidates for receiving intrapartum prophylaxis to prevent perinatal GBS infection, when compared with rectovaginal screening culture in the last trimester of gestation alone.