To determine the burden of illness caused by Escherichia coli O157 infections in populations in Foodborne Diseases Active Surveillance Network (FoodNet) surveillance areas, we initiated active, laboratory-based surveillance and surveyed laboratories, physicians, and the general public regarding the factors associated with the diagnosis and surveillance of infection with E. coli O157. We evaluated survey responses and site-specific incidence, outbreak, and demographic data during 1996-1999. A total of 1425 laboratory-confirmed cases of E. coli O157 infection and 32 outbreaks were reported from the 5 original FoodNet sites. The average annual incidence ranged from 0.5 cases/100,000 population in Georgia to 4.4 cases/100,000 population in Minnesota. After excluding outbreak-associated cases, the annual incidence of sporadic, laboratory-confirmed E. coli O157 infections remained relatively stable during 1996-1999, with a range of 1.9-2.3 cases/100,000 population. Regional differences in incidence partly resulted from differing physician and laboratory practices and from site-specific exposure factors (e.g., living on or visiting farms).