Whereas much information on the die-off of Escherichia coli in the aquatic environment is available, only few data support its growth under such conditions. We therefore investigated batch growth in microcosms containing different types of sterile freshwater. The water samples were inoculated with low starting cell concentrations of E. coli O157 (3 × 103 cells ml−1) and growth was followed using nucleic acid staining combined with flow cytometry. We demonstrated that E. coli O157 is able to grow in sterile freshwater at low carbon concentrations, which is against the common view that cell numbers decline over time when added to freshwater samples. A correlation between apparent assimilable organic carbon (AOCapp) concentration and the final cell concentration reached by E. coli O157 was established (P < 0.01). A considerable fraction of the AOCapp (34 ± 13%) was used by E. coli O157 but the numerical cell yield was about five-times lower in comparison with the bacterial AOC-test community, which originated from natural freshwater. On average, the maximum specific growth rate (μmax) of E. coli O157 growing in sterile freshwater at 30°C was 0.19 ± 0.07 h−1. Batch growth assays at five different temperatures revealed a positive influence of temperature on μmax of E. coli O157. The results give new information on the behaviour of this common pathogen in the aquatic environment and contribute to microbial risk assessment in order to prevent spreading of water-borne diseases.