During an investigation of chromosomal variation in strains of the ST239-MRSA-III clone, we identified and shotgun-sequenced a ~19 kb chromosomal insertion. Homologous sequences to this insertion were identified in the chromosomes of 6 S. aureus strains for which genome sequences are publicly available; however, these sequences were located at 5 different chromosomal loci in the different strains. Closer inspection of these sequences revealed that they were flanked by 3 bp direct repeats. We suspected that this insertion might encode a new mobile genetic element. Using PCR and Southern blotting, we detected the element in 12 different MLST-defined clones, indicating a wide distribution in the species. Moreover, Southern blotting suggested that some S. aureus strains contained multiple copies of the element. Using outward-directed PCR, cloning, and sequencing, we detected the presence of extrachromosomal circular forms of the element. The circular forms included 3 bp coupling sequences that corresponded to the 3 bp direct repeats flanking the chromosomal forms. The direct repeats were therefore interpreted as target-site duplications generated upon chromosomal integration of the element. In no case was a circular form detected in the absence of a chromosomal form, indicating that the element may not be a self-replicating plasmid. Various regions of the element were faintly homologous to components of bacterial conjugation systems including a putative relaxase, lytic transglycosylase, two coupling proteins, and a lipoprotein. These homologies indicated that the element might be self-transmissible and conjugative, rather than just mobilizable. Interestingly, faint homology to the IS30 family of DDE transposases was found where the tyrosine or serine recombinase of a typical conjugative transposon would be located. IS30 has been shown by other investigators to be capable of mediating site-specific recombinations in Escherichia coli. Thus, the IS30-like sequence might encode the recombinase for this element. This hypothesis is consistent with the fact that, in ST239 strains, the element contains a Tn552 insertion within the IS30-like sequence and we could not detect circular forms in these strains. Further analyses showed that the element had mosaic sequence characteristics, indicating a recombinational history. The element is of potential clinical relevance due to its variable carriage of Tn552, which confers penicillin resistance, and the observation that the element is a major source of gene content variation among strains of the USA300 clone. We classified this element, ICE6013, as an integrative and conjugative genetic element, reflecting its likely modes of maintenance and transmission.