BACKGROUND: Continuous monitoring systems have allowed determination of the time-to-positivity (TTP). We evaluated the clinical relevance of TTP in the BACTEC9240 system (Becton-Dickinson, USA). METHODS: A total of 2,354 vials of positive blood cultures were evaluated over 2 months. TTP was monitored from each of BACTEC Plus Aerobic/F (BD) or Pediatric Plus/F and Lytic Anaerobic/F bottles, and the differential time-to-positivity (DTP) for blood samples drawn simultaneously via catheter and a peripheral site was determined. RESULTS: The average TTP of the positive vials was 17.4 hr, and 79.9% and 95.2% of the vials showed positivity within 24 and 48 hr, respectively. While the average TTP values for Aeromonas hydrophila, Bacillus cereus, Acinetobacter baumannii, and Streptococcus pneumoniae were less than 10 hr, those for Candida spp., anaerobes, Propionibacterium acnes, Corynebacterium spp, Bacillus spp. other than cereus, and coagulase-negative staphylococci were 35.3, 27.0, 56.8, 45.8, 23.0, and 26.3 hr, respectively. The negative predictive values of TTP over 24 hr to predict Staphylococcus aureus among staphylococci and S. pneumoniae among alpha-hemolytic streptococci were 76.7% and 100%, respectively. Enterobacteriaceae and Enterococcus faecalis showed shorter TTP in anaerobic vials than in aerobic vials. DTP of more than 2 hr was observed for 27.8%, 72.2%, and 45.5% of S. aureus, S. epidermidis, and Candida spp. CONCLUSIONS: TTP can be used to discriminate pathogens and contaminants. The shorter TTP in anaerobic vials of certain Enterobacteriaceae and Enterococcus spp. would facilitate further identification. DTP is useful for diagnosing catheter-related bloodstream infection by S. aureus, S. epidermidis, and Candida spp.