Reduced affinity of penicillin-binding proteins (PBPs) for binding penicillin has been proposed as a mechanism of beta-lactam antibiotic resistance in staphylococci. Penicillin binding by PBPs of three penicillin-susceptible and two penicillin-resistant strains of Staphylococcus aureus was studied in kinetic assays to determine rate constants, drug concentrations at which PBPs were bound and the relationship between concentrations that bound PBPs and concentrations that inhibited bacterial growth. PBPs 1 and 2 of the resistant strains exhibited slower acylation and more rapid deacylation than susceptible strains. In contrast PBP 4, a naturally low-affinity PBP, was modified such that it exhibited a lower rate of deacylation. The concentrations of penicillin at which modified PBPs were bound correlated with concentrations that inhibited growth of the resistant strains. Acquisition of penicillin resistance in these strains of S. aureus results, at least in part, from structural modifications affecting binding of multiple PBPs and appears to include recruitment of a non-essential PBP, PBP 4.