Growth of methicillin-resistant Staphylococcus aureus DU4916 in the presence of methicillin yielded crude cell walls that showed an increased rate of autolysis and purified cell walls (PCW) and peptidoglycan (PG) that had increased susceptibilities to autolysin extracted with LiCl and to lysozyme. The PG of cells grown in the presence of methicillin had markedly decreased cross-linking and O acetylation. Growth of the methicillin-susceptible strain H in the presence of subinhibitory concentrations of cefoxitin, a specific inhibitor of penicillin-binding protein (PBP) 4, caused a substantial decrease in PG cross-linking and O acetylation and increased susceptibilities of PCW and PG to LiCl-extracted autolysin and to lysozyme. Strain DU4916 cells grown in the presence of methicillin did not show an increased rate of autolysis or an increased susceptibility to vancomycin- or D-cycloserine-induced lysis, even though their PG was hypo-cross-linked. This implies that the potential for increased autolysis is controlled in intact cells and that this regulation may be involved in the methicillin resistance phenomenon. Growth of the methicillin-susceptible strain DU4916S in the presence of methicillin yielded PCW and PG that showed small increases in susceptibilities to LiCl-extracted autolysin and to lysozyme and a small decrease in PG cross-linking. Comparison of the PBPs of a penicillinase-nonproducing derivative of strain DU4916 (DU4916-K7) with those of strain DU4916S in intact cells and isolated membranes revealed that PBPs 1 to 4 had similar high beta-lactam antibiotic affinities in both strains and identified an additional PBP, PBP2(1), with low beta-lactam affinity in the methicillin-resistant strain DU4916-K7. The low degree of cross-linking of PG in strain DU4916 cells grown with methicillin was probably due mainly to inhibition of the secondary cross-linking function of PBP 4.