We compared the pharmacodynamic activities of levofloxacin versus vancomycin, with or without rifampin, in an in vitro model with infected platelet-fibrin clots simulating vegetations. Infected platelet-fibrin clots were prepared with human cryoprecipitate, human platelets, calcium, thrombin, and approximately 10(9) CFU of organisms (MSSA 1199 and MRSA 494) per g and then were suspended via monofilament line into the in vitro model containing Mueller-Hinton growth medium. Antibiotics were administered by bolus injection into the model to simulate human pharmacokinetics; the regimens simulated included levofloxacin at dosages of 800 mg every 24 h (q24h) and 400 mg q12h, vancomycin at 1 g q12h, and rifampin at 600 mg q24h. Each model was run in duplicate over a 72-h period. Infected platelet-fibrin clots were removed in duplicate from each model, weighed, homogenized, serially diluted with sterile 0.9% saline, and plated on tryptic soy agar plates and plates containing antibiotics at 3, 6, and 12 times the MIC to evaluate the emergence of resistance. Time-kill curves were constructed by plotting the inoculum size versus time. Residual inoculum at 72 h was used to compare regimens. All levofloxacin regimens were significantly better than vancomycin monotherapy against both isolates (P < 0.002). Against MSSA 1199, levofloxacin q24h was significantly better than all other regimens, including levofloxacin q12h (P < 0.002); however, no difference between the levofloxacin monotherapy and combination therapy (with rifampin) regimens against MRSA 494 was seen. Killing activity for levofloxacin appeared to correlate better with the peak/MIC ratio than with the area under the curve/MIC ratio. The addition of rifampin significantly enhanced the activity of vancomycin but had little effect upon the activity of levofloxacin. For MRSA 494, vancomycin plus rifampin resulted in the greatest killing (P < 0.05). Development of resistance was not detected with any regimen. Levofloxacin may be a useful therapeutic alternative in the treatment of staphylococcal endocarditis, and further study with animal models of endocarditis or clinical trials are warranted.