Anorectal carriage as a possible primary source of vaginal colonization by group B Streptococcus was investigated. The study was performed during two separate periods and included 789 pregnant women and 422 neonates. Specimens from multiple sites were obtained for culture from all women and infants and were streaked onto blood agar plates containing 8 mug of gentamicin sulfate/ml and 15 mug of nalidixic acid/ml, which allow selective growth of streptococci. Cultures positive for group B streptococci were obtained from 162 (20.5%) of the pregnant women and from 50 (11.8%) of the neonates. Rectal cultures were positive for streptococci in 142 (17.9%) of the women, and vaginal cultures gave positive results in 81 (10.2%). The higher incidence of positive results in rectal as opposed to vaginal cultures (ratio of 2:1) was encountered during all phases of the study. This finding suggests that the gastrointestinal tract may be the primary site of colonization by group B Streptococcus and that vaginal colonization may represent contamination from this source.