Using a quantitative in vitro model simulating clinical conditions, we studied the efficacy of conventional and nonconventional 3-day therapies involving vancomycin for treating the internal surface of catheters colonized with a slime-producing strain of Staphylococcus epidermidis. When infused for 1 hr every 8 hr through the catheter at the daily dose recommended for a 10-kg child (450 mg), vancomycin alone reduced bacterial colonization but failed to sterilize the inserts. Vancomycin was more active in combination with netilmicin (25 mg for 1 hr every 8 hr), rifampin (150 mg for 90 min every 12 hr), or fosfomycin (500 mg for 4 hr every 6 hr), but the catheters were inconsistently decontaminated after 3 days of treatment. Two nonconventional modes of antibiotic administration were tested for their capacity to ensure high levels of vancomycin in the catheter lumen over a prolonged time. Vancomycin infused continuously through the catheter at a daily dose of 450 mg had the same poor sterilizing effect as vancomycin administered intermittently. On the contrary, catheters were totally decontaminated when 2.5 mg of vancomycin in a volume of 0.5 ml were injected twice daily into noninfused catheters, confirming that the antibiotic-lock technique is an approach of great interest to sterilize the internal surface of catheters colonized with staphylococci.