Infections associated with implants are frequently resistant to conventional antibiotic therapy. This resistance has been ascribed to the presence of bacteria on the artificial surface within a protective glue-like matrix forming a bacterial biofilm. We have demonstrated that experimental biofilms of Staphylococcus epidermidis, the main pathogen associated with implantation, are exquisitely sensitive to the action of rifampin. This effect of rifampin is incomplete, however, with the emergence of rifampin-resistant survivors that readily repopulate the biofilm. Studies were therefore performed to determine the effect of combinations of 13 different antibiotics with rifampin against standardized S. epidermidis biofilm preparations in an in vitro assay enabling the kinetic measurement of antibiotic action over a five-day period. The antibiotic combinations with rifampin demonstrated unsuspected divergent patterns of antimicrobial activity against the biofilms: 1. rapid synergy with rifampin was observed with cell-wall active antibiotics (cloxacillin, cephalothin, cefazolin, and cefamandole), whereas slower synergy occurred with vancomycin, ciprofloxacin, tetracycline, and amikacin; 2. some antibiotics (tobramycin, erythromycin, clindamycin, fusidic acid) did not influence the outcome; 3. gentamicin unexpectedly showed marked antagonism to rifampin. These results are relevant to the design of optimal therapeutic regimens for the management of resistant implant-associated infections.