Adult cancer patients were prospectively studied to determine the relationship between ultrastructural and microbiologic catheter colonization and clinical catheter-related infections. Participants included 38 patients whose central venous catheters were removed because of suspected catheter infection (16 patients) or other noninfectious causes (22 controls). The presence of clinical infection was determined. Catheters were examined by microbiologic methods (sonication and roll-plate culture) and by scanning and transmission electron microscopy. Ultrastructural microbial colonization and biofilm formation were universal and occurred as early as one day after catheter insertion. The extent of biofilm formation was unrelated to the clinical status of patient or the catheter microbiological findings. Secondary seeding of catheters was rarely seen. Catheter microbial biofilm formation occurs early, is universal and does not necessarily represent an infectious condition.