The aim of this study was to characterize the resistance mechanism in four clinical and five intestinal vancomycin-resistant Enterococcus faecium strains with VanB phenotype recovered from unrelated patients confined in two Spanish hospitals and to determine their clonal relationships. MIC values for vancomycin and teicoplanin were 16-32 and 0.5 microg ml-1, respectively. The mechanism of vancomycin resistance, as well as the genetic environment of the implicated gene, was analysed by PCR and sequencing. The vanB2 gene was detected in all nine E. faecium strains and the intergenic vanSB-YB region showed the characteristic mutations of the vanB2 subtype. Two possibly related PFGE patterns, A (seven strains) and B (two strains), were distinguished among these enterococci. The vanXB-ORFC intergenic region was amplified in the nine strains and two amino acid changes were detected in the protein encoded by the vanXB gene in strains of pattern A with respect to those of pattern B. The vanB2 gene cluster was integrated into Tn5382 in all nine strains, being pbp5 gene-linked to this transposon. The ant(6')-Ia, aph(3')-IIIa and erm(B) genes were also detected in all of the strains. Both isolates with PFGE pattern B contained the esp gene. In summary, vanB2-containing E. faecium strains with indistinguishable PFGE patterns were recovered from seven patients from two Spanish hospitals.