The emergence of clinical isolates of methicillin-resistantStaphylococcus aureus with reduced susceptibility to vancomycin has prompted a search for new and novel therapeutic agents active against S. aureus. Lysostaphin, a peptidase produced by Staphylococcus simulans, specifically cleaves the glycine-glycine bonds unique to the interpeptide cross-bridge of theS. aureus cell wall. The effectiveness of various regimens of dosing with intravenous lysostaphin was compared to that of vancomycin in the rabbit model of aortic valve endocarditis caused by a clinical methicillin-resistant S. aureus isolate. All animals were treated for a total of 3 days. The most active regimen, lysostaphin given three times daily, produced sterile vegetations in 10 of 11 treated rabbits, with a mean reduction in vegetation bacterial counts of 8.5 log₁₀ CFU/g compared to the counts in the untreated controls. In contrast, vancomycin given twice daily sterilized no vegetations and reduced vegetation bacterial counts by only 4.8 log₁₀ CFU/g. Lysostaphin given once daily was less effective, reducing mean vegetation bacterial counts by only 3.6 log₁₀ CFU/g, but the combination of lysostaphin once daily and vancomycin twice daily reduced the mean vegetation bacterial density by 7.5 log₁₀ CFU/g, a result that was significantly better than that for either regimen alone (P < 0.05). Lysostaphin was well tolerated by the rabbits, with no evidence of immunological reactions following up to 9 weeks of intravenous administration. We conclude that lysostaphin given alone or in combination with vancomycin is more effective in the treatment of experimental methicillin-resistant S. aureus aortic valve endocarditis than vancomycin alone.