We attempted to define the minimum blood volume and bacterial concentration required to obtain a positive blood culture with the use of placental blood and an in vitro technique. Known amounts of either Escherichia coli or group B beta-hemolytic streptococci were added to heparinized placental blood specimens. Blood samples of 0.25, 0.5, and 1.0 ml containing bacteria were inoculated into 30 ml of a commercially available broth culture medium, incubated for 24 hours, and examined for bacterial growth. Samples of at least 0.25 ml blood containing more than 10 colony-forming units of bacteria per milliliter resulted in a positive blood culture for 131 of 132 samples. On the basis of these data, we suggest that if 0.25 ml of blood is sampled and the specimen contains more than 10 colony-forming units per milliliter of E. coli or group B beta-hemolytic streptococci, the blood culture is almost certain to be positive.