Growth of two independently isolated strains of methicillin-resistant Staphylococcus aureus (MRSA) in increasing concentrations of methicillin (step-selection) resulted in increased resistance in these strains. When chromosomal DNA from the step-selected variants was probed using DNA sequences previously demonstrated to be associated with methicillin resistance in MRSA strains, amplification of the homologous chromosomal sequence was identified. Growth of these step-selected strains in the absence of methicillin resulted in loss of the amplified sequence, while the original sequence remained. There are differences between the two strains in the stability of maintenance of amplified sections. Prolonged storage of the variants on a high concentration of methicillin resulted in loss of amplified sections without concomitant loss of methicillin resistance. Thus amplification may be only one of at least two molecular mechanisms available to S. aureus to increase methicillin resistance in response to step-selection. Probing of cells of the highly resistant sub-population of a heterogeneously resistant MRSA strain showed that duplication of this mec-associated DNA is not involved in the mechanism of heteroresistance.