Acquired vancomycin resistance in enterococci may be associated with teicoplanin susceptibility (VanB) or teicoplanin resistance (VanA). This paper characterizes the first instance of in vivo emergence of teicoplanin resistance in an Enterococcus faecium strain of VanB phenotype. Vancomycin-resistant (MIC, 256/512 micrograms/mL) E. faecium was isolated intermittently from a patient's blood over 4 months. The MIC of teicoplanin for the first 5 isolates was 1.0 micrograms/mL; it was 64 micrograms/mL for the final 2. Analysis of plasmid and chromosomal DNA revealed the isolates to be of clonal origin. Conjugal transfer of vancomycin resistance was not obtained. A vanB DNA probe hybridized with both teicoplanin-susceptible and resistant isolates, but a vanA probe failed to hybridize with any isolate. SDS-PAGE of membrane proteins from a teicoplanin-resistant isolate revealed constitutive production of a normally inducible 41-kDa protein. These findings challenge the ultimate utility of teicoplanin for treatment of infections caused by vancomycin-resistant enterococci.