Thirty-six VanB glycopeptide-resistant Enterococcus faecium isolates were collected from patients in five different hospitals in Taiwan. The vancomycin resistance genes were amplified by the long vanB PCR, which amplifies the 6,373-bp vanB gene cluster including the vanR(B2), vanS(B2), vanY(B2), vanW(B2), vanH(B2), vanB2, and vanX(B2) genes. The deduced amino acid sequences were found to be 95 to 98% homologous to those of the vanB1 gene cluster: VanR(B1), 97%; VanS(B1), 97%; VanY(B1), 96%; VanH(B1), 95%; VanB1, 96%; and VanX(B1), 98%. Restriction enzyme analysis of the long vanB PCR products revealed that all 36 isolates had the same vanB2-specific pattern. DNA sequence analysis of the vanB2 gene, which is a D-Ala-D-Lac ligase gene, revealed that none of the 36 sequences were identical to the previously published vanB2 sequence. Thirty-one isolates had 1 nucleotide different from the published vanB2 sequence. The sequences of the other five isolates differed from the published vanB2 sequence by 2 or 3 nucleotides. Four isolates with a low or moderate resistance to vancomycin (MIC = 4 to 32 microg/ml) were found to have the same leucine-to-methionine change at amino acid position 308 of the vanB2 gene. The genomic DNAs of all 36 isolates were digested with SmaI and then typed by pulsed-field gel electrophoresis (PFGE). Eight different PFGE types (I to VIII) were observed, and type I was found to be prevalent in all hospitals examined in this study. This result suggests that intra- and interhospital dissemination of this E. faecium strain has occurred in Taiwan.