The aim of this study was to identify farm management factors associated with the prevalence of Escherichia coli O157:H7 among cattle in Ontario beef cow-calf operations. A total of 119 cow-calf operations with more than 50 cows in southern Ontario were visited between June and December 2002. From each farm, 65 fresh fecal samples were collected and cultured for E. coli O157:H7. Colonies of E. coli O157:H7 were isolated using immunomagnetic separation and standard microbiological techniques. Final confirmation of suspected colonies was based on identifying E. coli O157:H7-specific genes by PCR and serotyping of representative isolates. A questionnaire was administered to collect information on farm size, cattle demographics, farm management practices, the presence of other livestock and wildlife, and other aspects of the farm environment. Associations between the prevalence of E. coli O157:H7 in cattle feces and management factors were determined using a multivariable logistic regression model that included random effects for farm and county. The presence of pigs on farm, use of corn silage supplementation in winter, number of times cattle were taken to a show in the previous 12 months and the percentage of cows on farm were significant risk factors for the presence of E. coli O157:H7 in fecal pat samples, after controlling for region and the age group of the sampled animals. These findings highlight the potential roles of biosecurity and avoiding mixed animal agriculture in controlling the prevalence of E. coli O157:H7 in beef cow-calf operations.