Aims The objective of this study was to examine the prevalence of enteropathogenic Escherichia coli (EPEC) on beef and dairy farms and in beef abattoirs and to characterize the isolates in terms of serogroup and virulence markers. Methods and Results Bovine faecal samples (n = 1200), farm soil samples (n = 600), hide samples (n = 450) and carcass samples (n = 450) were collected from 20 farms and three abattoirs throughout Ireland over a 12-month period. After selective enrichment, samples testing positive for the intimin gene (eae) using PCR screening were cultured, and colonies were examined for the presence of the eae, vt1 and vt2 genes. Colonies that were positive for the intimin gene and negative for the verotoxin genes were further screened using PCR for a range of virulence factors including tir, espA, espB katP, espP, etpD, saa, sab, toxB, iha, lpfAO157/OI-141, lpfAO113 and lpfAO157/OI-154. PCR screening was also used to screen for variations in the intimin gene (eae). Of the 2700 source samples analysed, 3.9% (47 of 1200) of faecal, 2% (12 of 600) of soil, 6.4% (29 of 450) of hide and 0.7% (3 of 450) of carcass samples were PCR positive (for the presence of the eae gene). All 140 isolates obtained were atypical EPEC (aEPEC), while theta and beta intimin types were common. The virulence factors hlyA, tir, lpfA O113, lpfA O157/OI-154, and iha were frequently detected, while lpfAO157/OI-141, saa, espA, espB and toxB were also present but to a lesser extent. Conclusions It was concluded that cattle are a source of aEPEC, many of which have the virulence machinery necessary to be pathogenic to humans. Significance and Impact of the Study These findings suggest the need for increased research on aEPEC with particular emphasis on food safety and public health risk.