The development of freshwater multispecies biofilms at solid-liquid interfaces occurs both in quiescent waters and under conditions of high shear rates. However, the influence of hydrodynamic shear rates on bacterial biofilm diversity is poorly understood. We hypothesized that different shear rates would significantly influence biofilm diversity and alter the relative proportions of coaggregating and autoaggregating community isolates. In order to study this hypothesis, freshwater biofilms were developed at five shear rates (<0.1 to 305 S ؊1 ) in a rotating concentric cylinder reactor fed with untreated potable water. Eubacterial diversity was assessed by denaturing gradient gel electrophoresis (DGGE) and culturing on R2A agar. Fifty morphologically distinct biofilm strains and 16 planktonic strains were isolated by culturing and identified by partial 16S rRNA gene sequencing, and their relatedness was determined by the construction of a neighbor-joining phylogenetic tree. Phylogenetic and DGGE analyses showed an inverse relationship between shear rate and bacterial diversity. An in vitro aggregation assay was used to assess the relative proportions of coaggregating and autoaggregating species from each biofilm. The highest proportion of autoaggregating bacteria was present at high shear rates ( Microorganisms colonize a wide range of environments as spatially organized, taxonomically diverse, multispecies biofilm communities (1,20,44). In potable water distribution systems, many bacterial species, including members of the Proteobacteria, Actinobacteria, low-GϩC-content gram-positive bacteria, and Cytophaga-Flavobacterium-Bacterioides group, readily adhere to surfaces to form multispecies biofilms (29,41). The abilities of these taxonomically diverse organisms to attach to surfaces and codevelop within multispecies biofilms are imperative for their survival and persistence within flowing environments (4,33,44). Few published articles have described the effect of shear on the bacterial composition and diversity of freshwater biofilms. Evidence is emerging, however, that at high fluid velocities with associated high shear rates, multispecies communities are less diverse than those developed at lower shear rates (9,23,35,42). In order to adhere to surfaces or surface-attached cells and subsequently to form biofilms, bacteria in high-velocity flowing systems must overcome shear stress at the fluid-surface interface (7, 11). Attachment is facilitated by the expression of cell surface polymers that alter cell surface properties (3, 15). Properties that enhance attachment include increases in whole-cell hydrophobicity (14) and the abilities to coaggregate (25, 36) and autoaggregate (17). Recently, it was shown, using a simple recirculating tank model (35), that freshwater biofilms formed at high shear rates contained a high proportion of bacterial species that were not detectable in the surrounding fluid phase. In addition, a larger proportion of biofilm strains than of their planktonic counterparts were able to coaggr...