Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanS B / vanR B two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d -alanine: d -alanine ligase. Sequencing of the vanB operon in BM4525 also revealed an 18 bp deletion in the vanS B gene designated vanS . The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanS histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base-pairs. The VanS B , VanS and VanR B proteins were overproduced in Escherichia coli and purified. In vitro autophosphorylation of the VanS B and VanS histidine kinases and phosphotransfer to the VanR B response regulator did not differ significantly. However, VanS was deficient in VanR B phosphatase activity leading to accumulation of phosphorylated VanR B . Increased glycopeptide resistance in E. faecium BM4525 was therefore a result of the lack of production of d -alanyl- d -alanine ending pentapeptide and to constitutive synthesis of d -alanyl- d -lactate terminating peptidoglycan precursors, following loss of d -alanine: d -alanine ligase and of VanS B phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in vanS B may favour the appearance of high level constitutively expressed vancomycin resistance through a ‘slippage’ type of genetic rearrangement in VanB-type strains.