A ground beef patty processor detected Escherichia coli O157:H7 in five production lots during routine testing with polymerase chain reaction (PCR) technology. This finding stimulated research to determine the incidence and potential entry points of the pathogen during processing. One of these lots (53,960 kg) was divided into 71 pallets (760 kg each) of food service ground beef patties. Ten cartons (19 kg each) were removed from each pallet, for a total of 710 cartons. Four patties were taken from each carton and subdivided to provide comparable samples for E. coli O157:H7 analyses by three different laboratories. Two laboratories employed different immunoassay tests, and one used PCR to screen samples. One sample set was analyzed for aerobic plate, coliform, and E coli Biotype I counts to determine if any relationship existed between these microbial groups and the incidence of E. coli O157:H7. For 73 samples, presumptive positive results for E. coli O157:H7 were obtained by one or more methods. For 48 of these 73 samples, positive results for the pathogen were culture confirmed. The largest number (29) of culture-confirmed positive E. coli O157:H7 results were detected by PCR. Most positive results were obtained during a short segment of processing. All culture-confirmed E. coli O157:H7 strains were further characterized by two genetic subtyping techniques, resulting in two to four different patterns, depending on the subtyping procedure employed. For any sample tested, the aerobic plate count was < 3.0 log CFU/g, and coliform and E. coli Biotype I counts were < or = 1.00 log CFU/g. The results of this study suggest that most positive samples were associated with a contaminated batch of raw material introduced just before the 1725- to 1844-h processing segment. These results also indicate that more aggressive sampling plans and genetic screening technologies such as PCR may be used to better detect low levels of E. coli O157:H7 in ground beef products.