We describe a 4.5 kilobase transposon, Tn4001, which mediates resistance to gentamicin, tobramycin and kanamycin in Staphylococcus aureus. Originally detected in plasmid pSK1, Tn4001 was shown to undergo rec-independent transposition to the chromosome from this plasmid and from an inserted derivative of the plasmid pII147. Heteroduplexes between plasmids with and without Tn4001 demonstrated a characteristic stem and loop structure with inverted repeats of approx. 1.3 kilobases.