Current methods for diagnosis of catheter-related infection (CRI) are cumbersome and may require removal of the central venous catheter (CVC). A prospective study was conducted to validate the difference in time to detection (DTD) of cultures of blood samples obtained simultaneously from a peripheral vein (PV) and from the CVC for differentiation of CRI and non-CRI. During a 15-month period, 9 episodes were categorized as CRI and 24 as non-CRI. The median DTD for patients with CRI was significantly higher than that for patients with non-CRI (457 vs. -4 min; P<.001). The optimum cutoff point for diagnosis of CRI was a DTD of > or =120 min (sensitivity, 88.9%; specificity, 100%). With pretest probability of CRI ranging from 28% to 54%, the positive predictive value of a DTD of > or =120 min for the diagnosis of CRI was 100%; the negative predictive value was 89%-96%. On the basis of findings from this study, which is the largest, to date, to involve pediatric patients with tunneled CVCs and the first to use paired quantitative blood cultures as a "criterion standard," DTD was found to be a simple, reliable tool for diagnosis of CRI in hospitals that use continuously read blood culture systems.