We have identified a 26-kb mobile element from Enterococcus faecalis CH116, designated Tn5384, which confers resistance to erythromycin and to high levels of gentamicin. Tn5384 is a composite element containing three copies of insertion element IS256. Two of the IS256 copies flank the aac6'-aph2" bifunctional aminoglycoside-modifying-enzyme gene in the inverted orientation, forming a structure similar to staphylococcal gentamicin resistance transposon Tn4001. One of the IS256 elements involved in the Tn4001-like structure also forms the left end of Tn5384, the right end of which is a directly repeated insertion of IS256 approximately 23 kb downstream of the leftmost insertion. Insertions of Tn5384 into enterococcal plasmid pLRM1 have been found associated with 8- and 9-bp duplications of the target sequence.