Intimin or EaeA protein has been implicated in the attaching/effacing lesion caused by entero-hemorrhagic Escherichia coli (EHEC) in the intestine but it is not produced by all EHEC and is therefore not adequate as a marker for EHEC. Hemolysins are produced by a high percentage of verotoxigenic E. coli (VTEC) and could be a marker for EHEC, but their distribution and relation to virulence are not known. We used PCR amplification to determine the presence or absence of eaeA sequences in 281 VTEC isolates from the feces of healthy cattle. There were 101 eaeA-positive isolates, which belonged to O groups 5, 26, 69, 80, 84, 98, 103, 111, 119, 145, 157 and 108 eaeA-negative isolates, which belonged to O groups 8, 22, 38, 113, 119, 116, 132, 153, 156 or untypable. All isolates were tested for hemolysis on horse blood agar and on washed sheep blood agar. PCR amplification was used to test for EHEC hemolysin, Ehly1, Ehly2 and alpha-hemolysin D sequences. Among eaeA-positive isolates 98% were positive for EHEC hemolysin sequences and were hemolytic on washed sheep red blood cell agar; the corresponding percentage for eaeA-negative isolates was 36%. Ehly1 and Ehly2 sequences were present in only 11 isolates (O groups 26, 84, 119 and 132). None of the eaeA-positive and 13 of the eaeA-negative isolates (including all 11 isolates of O group 132) were positive for alpha-hemolysin D gene sequences by PCR and alpha-hemolysin production on horse blood agar. We conclude that since Ehly1, Ehly2 and alpha-hemolysin occur at low frequency among bovine VTEC and serotypes implicated in human disease they are unlikely to be significant virulence factors. In contrast, EHEC hemolysin was present in almost all eaeA-positive VTEC isolates and in approximately one-third the eaeA-negative ones; it may be both a virulence marker and virulence factor but further testing is required. The study identified isolates with all combinations of eaeA and EHEC hly, which may be useful for further testing.