The vanB2 operon encoding glycopeptide resistance is an integral part of the putative conjugative transposon Tn5382. Characterization of clinical glycopeptide resistant derivatives from an epidemic ampicillin-resistant Enterococcus faecium strain showed precise chromosomal or plasmid insertions of a vanB2-containing Tn5382-like element. Conjugative transposition of the Tn5382-like element was not demonstrated in retransfer studies.