To elucidate factors that contribute to the development of vancomycin resistance in methicillin-resistant Staphylococcus aureus (MRSA).Forty-nine MRSA isolates were subjected to passage selection with vancomycin to isolate mutants with reduced susceptibility to vancomycin. One mutant was chosen for detailed molecular and biochemical characterization.Five vancomycin-resistant mutants (vancomycin MICs, 6-12 mg/L) were obtained in vitro from five MRSA parent isolates. Upon acquisition of vancomycin resistance, all mutants showed a concomitant decrease in oxacillin resistance. In one particular MRSA strain, selection for vancomycin resistance repeatedly produced deletions and rearrangements, including loss of the mecA gene. Pleiotropic phenotypical changes, such as yellow pigment formation, loss of haemolysis, thickened cell wall, increased resistance to lysostaphin and reduced cell wall turnover were observed in this mutant.Acquisition of vancomycin resistance in one MRSA strain triggered mecA deletion suggesting that this deletion, coupled to other rearrangements and/or mutations, may be responsible for the increased vancomycin resistance phenotype.