Molecular analysis of 17 genomically unrelated clinical VanB-type vancomycin-resistant enterococcus isolates from hospital patients in Germany, Norway, Sweden, the United Kingdom and the United States revealed three subtypes of the vanB gene cluster-vanB1, vanB2, and vanB3-which was in accordance with previous subtyping of the ligase gene sequence. There was no correlation between vanB subtype and levels of vancomycin resistance. All strains studied carried a structurally conserved vanB gene cluster as shown by long-range PCR (long PCR) covering 5,959 bp of the published sequence in vanB1 strain V583. Restriction analysis of long PCR amplicons displayed one unique vanB1 pattern and a second vanB2- and vanB3-specific pattern. The vanSB-vanYB intergenic sequences with flanking coding regions were identical within each vanB subtype with one exception. A U.S. vanB2 isolate had a 789-bp enlargement of this region containing a putative open reading frame (ORF) with substantial homology to an ORF in the Clostridium perfringens IS1469 insertion element. The molecular heterogeneity within the vanB gene cluster has implications for the selection of PCR primers, as the primers must ensure detection of all vanB subtypes, and is of importance when considering reservoirs and dissemination of vanB resistance. The molecular identity within the vanB1 and the vanB2 subtype indicates horizontal transmission of both gene clusters between isolates in different geographical areas. Restriction analysis of long PCR vanB amplicons may reveal specific varieties that can be used as epidemiological markers for mobile determinants conferring VanB-type resistance. The finding of three distinct vanB gene clusters should encourage a search for different environmental reservoirs of vanB resistance determinants.