In five vancomycin-resistant laboratory step mutants selected from the highly and homogeneously methi-cillin-resistant Staphylococcus aureus strain COL (MIC of methicillin, 800 mg/ml; MIC of vancomycin, 1.5 mg/ml), the gradually increasing levels of resistance to vancomycin were accompanied by parallel decreases in the levels of methicillin resistance and abnormalities in cell wall metabolism. The latter included a gradual reduction in the proportion of highly cross-linked muropeptide species in peptidoglycan, down-regulation of the production of penicillin-binding protein 2A (PBP2A) and PBP4, and hypersensitivity to b-lactam antibi-otics each with a relatively selective affinity for the various staphylococcal PBPs; the PBP2-specific inhibitor ceftizoxime was particularly effective. The mechanism of resistance in the recently described gly-copeptide-resistant clinical isolates of Staphylococcus aureus is not well understood. A laboratory mutant, VM50 (formerly called VM), with high-level resistance to vancomycin (MIC, 100 mg/ml) and selected from the methicillin-resistant S. aureus strain COL through several steps of exposure to vancomycin, showed several unusual properties. The most striking was a