MEASUREMENTS AND MAIN RESULTS: In an intensive care unit population, we prospectively studied the incidence of central venous catheter infection and colonization at the subclavian, internal jugular, and femoral sites. The optimal insertion site for each individual patient was selected by experienced intensive care physicians (critical care medicine attendings and fellows). All of the operators were proficient in inserting catheters at all three sites. Confounding factors were eliminated; there were a limited number of experienced operators inserting the catheters, a uniform protocol stressing strict sterile insertion was enforced, and standardized continuous catheter care was provided by dedicated intensive care nurses proficient in all aspects of central venous catheter care.Two groups of patients were analyzed. Group 1 was patients with one catheter at one site, and group 2 was patients with catheters at multiple sites. Group 1 was the primary analysis, whereas group 2 was supporting.A total of 831 central venous catheters and 4,735 catheter days in 657 patients were studied. The incidence of catheter infection (4.01/1,000 catheter days, 2.29% catheters) and colonization (5.07/1,000 catheter days, 2.89% catheters) was low overall.In group 1, the incidence of infection was subclavian: 0.881 infections/1,000 catheter days (0.45%), internal jugular: 0/1,000 (0%), and femoral: 2.98/1,000 (1.44%; p = .2635). The incidence of colonization was subclavian: 0.881 colonization/1,000 catheter days (0.45%), internal jugular: 2.00/1,000 (1.05%), and femoral: 5.96/1,000 (2.88%, p = .1338). There was no statistically significant difference in the incidence of infection and colonization or duration of catheters (p = .8907) among the insertion sites.