Although several reports have shown that two to three 20-ml blood cultures are adequate for the detection of bacteremia and fungemia in adults, a recent study (F. R. Cockerill et al., Clin. Infect. Dis. 38:1724-1730, 2004) found that two blood cultures detected only 80% of bloodstream infections and that three blood cultures detected 96% of episodes. We reviewed data at two university hospitals to determine whether the recent observations by Cockerill et al. are applicable more widely. We assessed all blood cultures obtained from adult inpatients from 1 January 2004 through 31 December 2005 at Robert Wood Johnson University Hospital and Duke University Medical Center. All instances in which > or =3 blood cultures per patient were obtained during a 24-h period were included. The medical records of patients who met the inclusion criteria were reviewed retrospectively to determine the clinical significance of the positive blood culture (true infection versus contamination). Data were analyzed to determine the cumulative sensitivity of blood cultures obtained sequentially during the 24-h time period. Of 629 unimicrobial episodes with > or =3 blood cultures obtained during the 24-h period, 460 (73.1%) were detected with the first blood culture, 564 (89.7%) were detected with the first two blood cultures, 618 (98.2%) were detected with the first three blood cultures, and 628 (99.8%) were detected with the first four blood cultures. Of 351 unimicrobial episodes with > or =4 blood cultures obtained during the 24-h period, 257 (73.2%) were detected with the first blood culture, 308 (93.9%) were detected with the first two blood cultures, 340 (96.9%) were detected with the first three blood cultures, and 350 (99.7%) were detected with the first four blood cultures. Among unimicrobial episodes, Staphylococcus aureus was more likely to be detected with the first blood culture (approximately 90% detected with the first blood culture). There were 58 polymicrobial episodes in which > or =3 blood cultures were obtained. Forty-seven (81.0%) were detected with the first blood culture, 54 (93.1%) were detected with the first two blood cultures, and 58 (100%) were detected with the first three blood cultures. The results of this study indicate that two blood cultures in a 24-h period will detect approximately 90% of bloodstream infections in adults. To achieve a detection rate of >99%, as many as four blood cultures may be needed. The previously held axiom that virtually all bloodstream infections can be detected with two to three blood cultures may no longer be valid but may also depend on the definition of the "first" blood culture obtained (see Materials and Methods and Discussion in the text).