All clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) examined so far contain the mec gene and its product, the penicillin-binding protein (PBP) 2A. Yet the same strains show tremendous variation in the phenotypic expression of antibiotic resistance (MIC), which is under the control of a set of additional, auxiliary genes. Thus, the quantitative contribution of the mec gene to the resistance phenotype of MRSA is not known, and no mutants with the lesion located within the mec gene have been described. We subjected a highly resistant MRSA strain to transposon mutagenesis with the erythromycin resistance transposon Tn551, and a mutant expressing greatly decreased methicillin resistance (RUSA4) was selected to characterize the transposon insertion site. The results indicate that the Tn551 insertion site in mutant RUSA4 is between base pairs 1000 and 1400 of the sequence encoding PBP 2A. Thus, the uniform and greater than 200-fold drop in the methicillin MIC (4 micrograms/ml) for this mutant relative to that for the parent strain (MIC greater than or equal to 800 micrograms/ml) must be related to the inactivation of the PBP 2A gene. The results provide the first unequivocal evidence for the importance of PBP 2A as a quantitative contributor to the MIC for MRSA.