Escherichia coli 0157 is a strain of enteric bacteria that is associated with two potentially fatal diseases, haemorrhagic colitis and haemolytic uraemic syndrome. The bacterium has been implicated with outbreaks of disease where the individual has been exposed either to water or to land contaminated with faecal material, or to food poisoning pertaining to inadequate slaughter, cooking, or hygiene practices. E. coli 0157 has been isolated from private and municipal water supplies, mainly as a result of contamination of the source water. Animal grazing and the use of untreated, manure-based fertiliser can lead to an influx of faecal bacteria to the soil, where cultivation and natural run-off transfer the bacteria deep into the soil, as well as ground and surface waters. A dairy farm in East Sussex (United Kingdom) was used to investigate the potential of virulent E. coli 0157 isolates to exist within temperate freshwater biofilms. Devices using indigenous flint shingle were set up at four sites along the stream network to allow natural biofilm formation. Stones were removed from each site at four week intervals for one year, and screened to characterise eight Enterobacteriaceae genera and obtain total heterotrophic counts. Pooled faecal samples were obtained from indigenous grazing animals were also characterised to investigate their potential role as vectors of this pathogenic organism. A total of 1002 E. coli isolates were recovered from both the biofilm and animal faeces, 48 of which were identified as E. coli 0157. All E. coli isolates were analysed for kinetic biochemical phenotypes using the PhenePlateTM technique, and all confirmed E. coli 0157 isolates were subjected to an antibiotic resistance screen in an attempt to develop a host-specific resistance profile using the VetMICTM technique. The presence of five virulence traits known to cause or to be associated with human enteric disease were identified among confirmed E. coli O157 isolates using PCR. Phenotypic analysis revealed that distinct sub-populations of E. coli exist for each animal population, some of which displayed a significant phenotypic similarity to those recovered from the biofilms, suggesting that a source of faecal bacteria may be entering the streams. The antibiotic screen was not able to determine host-specific antibiotic profiles due to the small number of isolates recovered from the pooled faeces from each animal population (fourteen from pooled goat faeces, four from cattle, two from chickens and two from pigs). PCR confirmed the presence of the two verotoxin genes, those for intimin and an enterohaemolysin-encoding plasmid, as well as two ERIC sequences determined from E. coli O157 strains known to cause disease. This work supports research revealing that the bacterium can survive in environmental conditions that are in stark contrast to those of the mammalian gastro-intestinal tract. It suggests a link between strains that are excreted by grazing animals and those found in the aquatic epilithic biofilms, and represent a potential reservoir for the dissemination of disease to susceptible human populations. This research has implications for epidemiological studies, and suggests that disease control measures should be adjusted to account for the transfer of bacteria from land-based environments to aquatic ones, accounting for both natural and human-associated processes. E. coli 0157 is a clear and present danger to human health if ingested, but the bacterium exists in the biofilms as a naturallyoccurring organism, and should be treated as a result of natural run-off and leaching processes rather than the deliberate contamination of surface waters.