The vancomycin resistance vanB2 gene cluster is disseminated worldwide and has been found in phylogenetically remote bacterial genera. The vanB2 operon is part of conjugative transposons Tn1549/Tn5382, but conjugative transposition of these elements has not been demonstrated. We have obtained transfer of a Tn1549-like element (referred to herein as "Tn1549-like") from Clostridium symbiosum MLG101 to Enterococcus faecium 64/3 and Enterococcus faecalis JH2-2 in the digestive tract of gnotobiotic mice and to E. faecium 64/3 in vitro. Retransfer of Tn1549-like from an E. faecium transconjugant also containing Tn916 to E. faecium BM77 was obtained in vitro, albeit at a very low frequency. Transfer efficiency was found to be both donor and recipient dependent. Pulsed-field gel electrophoresis analysis of total SmaI-digested DNA of 48 transconjugants indicated in 27 instances the acquisition of ca. 34 kb of DNA. Two transconjugants harbored two copies of the transposon. Sequencing of the flanking regions of Tn1549-like in 48 transconjugants revealed 29 integration events in 26 loci in the E. faecium genome, and two hot spots for insertion were identified. Integration of the transposon was associated with the acquisition of 5 (n = 18) or 6 (n = 7) bp of donor DNA or with 5-bp duplications of target DNA in the remaining transconjugants. These data demonstrate functionality of the Tn1549-like element and attest that the transfer of the vanB operon between enterococci and human commensal anaerobes occurs in the intestinal environment.