To assess the degree of luminal and extraluminal colonization of long-term central venous catheters (CVC), 359 indwelling silicone CVC from 340 consecutive cancer patients were examined. All CVC were cultured by the roll-plate and sonication quantitative culture techniques. Semiquantitative electron microscopy was done on 39 CVC associated with catheter infections and on 26 culture-negative controls. An additional 10 culture-negative CVC obtained after death were also studied by electron microscopy. Ultrastructural colonization and biofilm formation was universal and quantitatively independent of clinical catheter-related infections. Ultrastructural colonization and biofilm formation was predominantly luminal in long-term CVC (> 30 days). Based on a composite definition, the sensitivity of the roll-plate catheter tip culture was 42%-45% compared with 65%-72% for the sonication of the tip. Colonization of indwelling catheters is universal regardless of culture results. For long-term CVC, colonization becomes predominantly luminal and extraluminal quantitative catheter cultures are of limited diagnostic sensitivity.