Soon after methicillin was introduced into clinical practice in the early 1960s, resistant strains of Staphylococcus aureus (MRSA) appeared, bearing a newly acquired resistance gene, mecA, that encodes a penicillin binding protein, PBP2a. MRSA have spread throughout the world, and an investigation of the clonality of 472 isolates by DNA hybridization was performed. All 472 isolates could be divided into six temporally ordered mecA hybridization patterns, and three of these were subdivided by the chromomosomal transposon Tn554. Each Tn554 pattern occurred in association with one and only one mecA pattern, suggesting that mecA divergence preceded the acquisition of Tn554 in all cases and therefore that mecA may have been acquired just once by S. aureus.