We studied 21 women undergoing elective laparoscopy to determine whether positive bacterial cultures obtained by culdocentesis were contaminants from the vaginal mucosa. Cultures for aerobic and anaerobic microorganisms were prepared from swabs of the posterior vaginal fornix, laparoscopic cul-de-sac aspirates, culdocentesis aspirates, and repeat laparoscopic aspirates of the cul-de-sac. Cultures were positive from the vaginal fornix in all cases. All cultures from the cul-de-sac before culdocentesis were negative. Five of seven cultures (71.4%) from culdocentesis specimens and seven of nine cultures (77.8%) from repeat laparoscopic cul-de-sac aspirates were positive in women without povidone-iodine vaginal preparation. Five of 12 cultures (41.7%) from culdocentesis specimens and three of 12 cultures (25%) from repeat laparoscopic cul-de-sac aspirates were positive in the women with vaginal preparation. Both aerobic and anaerobic bacteria were recovered. We conclude that false-positive culdocentesis cultures occur and that bacterial contamination of the cul-de-sac following culdocentesis may lead to false-positive cultures of specimens obtained during subsequent laparoscopy.