The Staphylococcus aureus plasmid pSK1 carries Tn4001, a 4.7-kilobase (kb) transposon which specifies resistance to gentamicin, tobramycin, and kanamycin. In addition, pSK1 mediates resistance to trimethoprim and linked resistance to ethidium bromide (Eb(r)) and to quaternary ammonium compounds (Qa(r)). Restriction endonuclease analysis of pSK1 and a deleted derivative of pSK1 revealed that the gene(s) responsible for Eb(r) Qa(r) lies within a 5.2-kb HindIII fragment. This fragment has been cloned into the Escherichia coli plasmid vector pBR322, and transformants of an E. coli K-12 strain exhibited Eb(r) Qa(r). Subcloning of the 5.2-kb insert, combined with data from electron microscopic analysis of deleted derivatives of pSK1, located the Eb(r) Qa(r) determinant(s) on a 2.3-kb segment of pSK1 DNA.