The conjugation system of Staphylococcus aureus that is conferred, at least in part, by plasmid pJE1, requires cell-to-cell contact. Optimum transfer was found when the numbers of donor and recipient cells were equal. Certain antibiotics increased the conjugation frequency. Fragments of plasmid pJE1 were cloned into a staphylococcal plasmid vector; although separate clones were isolated that conferred ethidium bromide resistance and gentamicin resistance, none of the clones carried the ability to conjugate. Transposon mutagenesis with Tn551 was used to create 26 mutants of pJE1. These were analysed for the position of the insertion and for their ability to conjugate. The sites of insertion were non-random. Only six mutants were unaffected in their conjugability; one showed increased ability to conjugate whilst the rest were either unable to conjugate or showed a reduced frequency. It is concluded that there are at least two separate regions necessary for conjugation and that the system is not obviously similar to that reported in streptococci.