In the present study, the diversity and the phylogenetic affiliation of bacteria in a biofouling layer on reverse osmosis (RO) membranes were determined. Fresh surface water was used as a feed in a membrane-based water purification process. Total DNA was extracted from attached cells from feed spacer, RO membrane and product spacer. Universal primers were used to amplify the bacterial 16S rRNA genes. The biofilm community was analysed by 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) and the phylogenetic affiliation was determined by sequence analyses of individual 16S rDNA clones. Using this approach, we found that five distinct bacterial genotypes (Sphingomonas, Beta proteobacterium, Flavobacterium, Nitrosomonas and Sphingobacterium) were dominant genera on surfaces of fouled RO membranes. Moreover, the finding that all five "key players" could be recovered from the cartridge filters of this RO system, which cartridge filters are positioned before the RO membrane, together with literature information where these bacteria are normally encountered, suggests that these microorganisms originate from the feed water rather than from the RO system itself, and represent the fresh water bacteria present in the feed water, despite the fact that the feed water passes an ultrafiltration (UF) membrane (pore size approximately 40 nm), which is able to remove microorganisms to a large extent.