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This study aimed to analyse the diversity of the vanB gene cluster in enterococcal species isolated from sewage treatment plants (STP) in Tehran, Iran.The enterococcal isolates were collected from three sewage treatment plants in Tehran, Iran, during 2005. A total of 203 enterococcal isolates, collected over six rounds of sampling from three STPs were tested for the presence of vanB gene. Long-PCR showed that amongst the isolates, three Enterococcus faecium, one Enterococcus gallinarum and one Enterococcus casseliflavus harboured the complete vanB gene cluster.Restriction fragment length polymorphism (RFLP) of the vanB1 gene cluster (5900 bp) from the isolates showed an identical pattern to a standard strain of Enterococcus faecalis (V583). None of the isolates were able to transfer the vanB gene in conjugation experiments. Different pulsed-field gel electrophoresis patterns were obtained for the three E. faecium isolates with vanB gene clusters.Our results indicated that the dissemination of vanB is not widespread in Tehran. Although only a few vanB positive isolates were detected, vanB was found in several enterococcal species.In view of the lack of information on vanB resistance genes and their diversity in Iran, knowledge of the global dissemination of vanB genes in Enterococcus spp. is noteworthy.