Hexachlorobenzene (HCB) has been widely used in chemical manufacturing processes and as a pesticide. Due to its resistance to biological degradation, HCB has mainly accumulated in freshwater bodies and agricultural soils. "Dehalococcoides" spp., anaerobic dechlorinating bacteria that are capable of degrading HCB, were previously isolated from river sediments. Yet there is limited knowledge about the abundance, diversity, and activity of this genus in the environment. This study focused on the molecular analysis of the composition and abundance of active Dehalococcoides spp. in HCB-contaminated European river basins. 16S rRNA-based real-time quantitative PCR and denaturing gradient gel electrophoresis in combination with multivariate statistics were applied. Moreover, a functional gene array was used to determine reductive dehalogenase (rdh) gene diversity. Spatial and temporal fluctuations were observed not only in the abundance of Dehalococcoides spp. but also in the composition of the populations and rdh gene diversity. Multivariate statistics revealed that Dehalococcoides sp. abundance is primarily affected by spatial differences, whereas species composition is under the influence of several environmental parameters, such as seasonal changes, total organic carbon and/or nitrogen content, and HCB contamination. This study provides new insight into the natural occurrence and dynamics of active Dehalococcoides spp. in HCB-contaminated river basins.Halogenated organic compounds are among the most widespread environmental pollutants. Although these compounds were previously believed to be only anthropogenic, a large number of them, including aliphatic, aromatic, and heterocyclic derivatives, are introduced into the environment via biogenic and geogenic sources (9, 21). Hexachlorobenzene (HCB) is believed to be persistent in the environment (22) due to its chemical stability and its resistance to biodegradation. HCB is a hydrophobic and bioaccumulative compound and is listed in the EC Directive (15) as a "priority hazardous substance." At the peak production of HCB in the early 1980s, thousands of tons were produced to be used as fungicides, wood preservatives, and porosity control agents or in the manufacturing of dyes. The use of HCB is no longer allowed in most countries because of its toxicity and carcinogenicity toward fish and mammals. Nevertheless, it is still being released into the environment as a by-product of various chemical processes, as a result of incomplete combustion, or from old landfills (4, 6, 7). HCB contamination has been reported in different environments. Compared to rivers in sparsely populated regions, lakes, and the sea (32, 42), significantly larger amounts of HCB could be found in river water in agricultural areas and in densely populated or highly industrialized areas. HCB concentrations were shown to positively correlate with organic matter content of sediments and soils, and European soils were observed to have the highest HCB concentrations globally (38). Several authors reported o...