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Objective: To determine the duration of antimicrobial activity and the efficacy of indwelling catheters coated with minocycline and rifampin in preventing ultrastructural colonization., Design: Multicenter, prospective, randomized, clinical trial., Setting: Five university-based medical centers., Patients: Cohort 1 consisted of 40 randomized patients in whom an equal number of minocycline- and rifampin-coated and uncoated catheters were inserted and studied using scanning electron microscopy. Cohort 2 consisted of 118 patients who received coated catheters that were tested for the antimicrobial activity and levels of the antibiotics at the time of removal., Interventions: Catheters pretreated with tridodecylmethylammonium chloride and subsequently coated with minocycline and rifampin; uncoated catheters (control)., Measurements and Main Results: Quantitative scanning electron microscopy was utilized to determine both the ultrastructural colonization in biofilm on coated and uncoated catheters. The zones of inhibition of coated catheters from studied patients against Staphylococcus epidermidis was used to determine the antimicrobial durability. High-performance liquid chromatography was used to determine antibiotic levels on indwelling coated catheters and in serum. Mild-to-heavy ultrastructural colonization was detected in 7 (35%) of 20 coated catheters and in 16 (80%) of 20 uncoated catheters (p = .004). Significant antimicrobial inhibitory activity against S. epidermidis was maintained for 16 days. Rifampin and minocycline continued to be detected on the surfaces of coated catheters for at least 2 wks after placement. Neither antibiotic was detected in the 60 serum samples obtained from 15 patients during catheterization., Conclusion: Coating catheters with minocycline and rifampin inhibits ultrastructural colonization of indwelling catheters and maintains effective antimicrobial activity for at least 2 wks.