A prospective study of 45 central venous catheters was conducted to assess, by strain delineation, the turnover of skin and catheter hub (superficial) colonization and the relative contributions of catheter hub and skin colonization to catheter tip colonization. Serial quantitative cultures of skin and catheter hub were performed. Catheter tip, blood, and specimens for culture from targeted superficial sites (TSSs) were also collected at the time of catheter removal. Strains from 17 tip-positive catheters were delineated by pulsed-field gel electrophoresis. Only 12 (28.6%) of 42 skin strains and 14 (31.1%) of 45 catheter hub strains were found to be present at the time of catheter removal. In addition, only 9 (29.0%) of the 31 tip-colonizing strains were present on TSSs. Moreover, 15 (48.4%) of the 31 tip-colonizing strains had a superficial origin, and the other 16 (51.6%) were of unknown origin. In catheters suspected of infection, cultures of TSSs had a negative predictive value for catheter-related bacteremia of 94.4% but a positive predictive value of 44.4%. When the causative agent was identified (to the strain level) these values dropped to 80.9 and 18.7%, respectively. The study shows that skin and catheter hub colonization is a common, dynamic phenomenon. Strains recovered from TSSs showed a low level of correlation with strains from previous cultures of specimens from superficial sites and catheter tip isolates. Consequently, TSSs cannot be recommended for use in determining the therapy. However, catheter-related bacteremia is uncommon when cultures of TSSs are negative.