In a previous study, the seasonal prevalence was reported for stx+ Escherichia coli O157:H7 in feces and on hides and carcasses of cattle at processing. Overall, 1,697 O157:H7 isolates have now been characterized for the incidence of (i) eae(O157), hlyA, stx1, and stx2 in the recovered isolates and (ii) presumptive rough and presumptive nonmotile isolates. Seven O157:H7 isolates (0.4%) lacked stx genes, although they carried eae and hlyA. All but one of the isolates carried both eae and hlyA. Approximately two-thirds of the isolates (64% when one isolate per sample was considered) carried both stx1 and stx2. E. coli O157:H7 cells that harbored both stx1 and stx2 were more often recovered from hides in the fall (79% of the fall hide isolates) and winter (84% of the winter hide isolates) than in the spring (53%) and summer (59%). Isolates recovered from preevisceration carcasses showed a similar but not statistically significant trend. Twenty-three of the 25 O157:H7 isolates carrying stx1 but not stx2 were recovered during summer. Fifteen presumptive rough and 117 presumptive nonmotile stx+ O157:H7 isolates were recovered. Ten (67%) of the presumptive rough isolates were recovered during summer. Ninety-five of the presumptive nonmotile isolates (81%) were recovered during fall. Forty-eight percent of the false-positive isolates (175 of 363) tentatively identified as O157:H7 were O157+ H7- and lacked eae(O157), hlyA, and stx. These data suggest that in beef processing samples (i) there are minor seasonal variations in the prevalence of stx genes among E. coli O157:H7 isolates, (ii) presumptive rough and presumptive nonmotile stx+ O157:H7 isolates are present, (iii) E. coli O157:H7 isolates lacking stx genes may be rare, and (iv) O157+ H7- isolates lacking stx genes can result in many false-positive results.