OBJECTIVE Diagnosis of neonatal catheter-related bloodstream infection (CRBSI) is currently based on isolation of identical bacterial species from bloodstream and catheter tip cultures. This requires removal of the catheter followed by the insertion of a new catheter. The objective of this study was to investigate whether differential time to positivity (DTP) of blood cultures drawn from paired peripheral vein and central vascular catheter is useful for diagnosing neonatal CRBSI, avoiding removal of the catheter. DESIGN Retrospective observational study. SETTING Neonatal intensive care unit, University Hospital of Antwerp, Belgium. PATIENTS Neonates with probable and definite nosocomial bloodstream infection. INTERVENTIONS All episodes of nosocomial bloodstream infection (NBSI) in an approximately 7.5-yr period were identified retrospectively. Definite NBSI episodes in which paired blood cultures were obtained were retained to calculate DTP, to determine the optimal DTP cutoff for the diagnosis of CRBSI, and to assess the validity of DTP for the diagnosis of CRBSI. MEASUREMENTS AND MAIN RESULTS Of 32 NBSI episodes included in the study, 16 were CRBSI, seven were non-CRBSI, and nine were classified as "diagnosis uncertain." In CRBSI, blood cultures drawn from a central vascular catheter were positive earlier than those drawn from a peripheral vein (median 9.67 hrs vs. 21.58 hrs, p < .01). Median DTP was 10.42 hrs in CRBSI and -0.33 hrs in non-CRBSI (p = .01). The optimal DTP cutoff for the diagnosis of CRBSI was > or =1 hr (area under the receiver operating characteristic curve = 0.84 +/- 0.11), with a sensitivity of 94%, a specificity of 71%, a positive predictive value of 88%, and a negative predictive value of 83%. CONCLUSIONS Differential time to positivity of paired blood cultures may have some potential in the diagnosis of catheter-related infections in neonatal intensive care unit patients and should be subjected to a prospective study.