Levels of rifampin, gentamicin, sisomicin, and cephalothin in normal and osteomyelitic rabbit bones were measured, and the efficacy of these drugs in the treatment of osteomyelitis was evaluated. Single drug regimens, including rifampin for 14 days and gentamicin, sisomicin, and cephalothin each for 28 days, were relatively ineffective (5%-33% sterile bone cultures). Rifampin, administered for 28 days, sterilized the bones of 55% of treated animals. The combination of gentamicin and rifampin, given for either 14 or 28 days, sterilized the bones of 67% of treated animals. The combinations of rifampin plus sisomicin and of rifampin plus cephalothin, given for 28 days, were significantly more effective than these agents alone, sterilizing 90%-95% of bones. The combination of rifampin, sisomicin, and cephalothin, given for only 14 days, sterilized the bones of all treated rabbits. Staphylococci isolated from the bones of therapeutic failures that had received rifampin alone or in combination with other antibiotics were highly resistant to rifampin (minimal inhibitory concentration, greater than 250 mug/ml), whereas the organisms recovered from animals not receiving rifampin remained sensitive. Results of in vitro studies of synergy and/or bactericidal activity of antibiotic combinations correlated with in vivo results in some, but not all, instances.