The restriction site polymorphism of the chromosomal femAB region and the first appearance of the regulatory element mecR1-mecI associated with the methicillin resistance determinant (mec) were analyzed in 192 initially methicillin resistant (Mcr) Staphylococcus aureus clinical isolates collected between 1965 and 1990 in the Zurich area. Forty-three of the strains lost the resistance spontaneously. All isolates that were still Mcr hybridized with mecA, the gene for the low-affinity penicillin-binding protein PBP 2'. Mcr strains isolated before 1977 lacked sequences that hybridized with mecR1-mecI, a regulatory element controlling the expression of mecA; exceptions to this were one strain isolated in 1966 and one strain isolated in 1972. The size of the EcoRV fragment carrying femA, a chromosomally encoded factor involved in pentaglycine side chain formation of the peptidoglycan and essential for the expression of methicillin resistance, was conserved in all strains but one, which was susceptible to methicillin even though it carried a functional mecA gene. The methicillin susceptibility of this particular strain was presumably due to a spontaneous femA-like mutation. The 192 strains belonged to seven different EcoRV restriction fragment patterns recognizable with a 10.5-kb probe covering the femAB region. Some 93% of the 149 Mcr strains belonged to pattern A, and the remaining Mcr strains shared patterns A' and B. The 42 isolates which spontaneously lost their resistance upon storage and revival represented all seven different patterns. This strong conservation of femA suggests an important role for femA in cell wall metabolism and methicillin resistance.