The accumulation of infected clots under the silicone septum of the reservoir of venous access ports (VAPs) has been reported. We analyzed the relationship between these deposits and the occurrence of VAP-related bloodstream infections (VAP-BSIs) by (1) evaluating the accuracy of paired quantitative blood cultures for diagnosing VAP-BSI before the removal of the device and (2) assessing the accuracy of cultures of the tip and septum (i.e., the internal lumen of the VAP) for diagnosing VAP-BSI after removal of the device. Over a 16-month period, all VAPs removed were prospectively investigated. Before VAP removal, paired quantitative blood cultures were 77% sensitive and 100% specific and had a positive predictive value of 100% and a negative predictive value of 98% for diagnosing VAP-BSI. After VAP removal, tip culture was only 46% sensitive, whereas septum culture was 93.3% sensitive for confirming the diagnosis of VAP-BSI. Thus infected deposits that accumulate under the VAP septum are the source of VAP-BSI.