A total of 90 Escherichia coli O157 isolates recovered from humans, cattle, and pigs, were examined for the presence of the H7 antigen, ability to produce Shiga toxins and enterohemolysin as well as for antimicrobial resistance and biochemical properties. Fourteen of the human strains (n = 23) and 21 of the bovine isolates (n = 29) were of the O157:H7 serotype as determined by agglutination and PCR methods. All E. coli O157 of porcine origin (n = 38) were H-negative. Based on the ability to produce Shiga toxins (Stxs), all human and cattle isolates and 11 strains recovered from swine were identified as Shiga toxin-producing E. coli (STEC). Among STEC, most human strains (18 isolates) were Stx1- and Stx2-positive whereas cattle strains were mostly Stx2-positive. Eleven porcine STEC produced either Stx1 (7 isolates) or Stx2 (4 strains) toxins; an additional 20 isolates recovered from these animals had the Stx2e toxin gene as previously determined by PCR. All human and cattle E. coli O157 produced enterohemolysin whereas only 4 strains recovered from pigs were ehly-positive. Moreover, the PCR identification of the lpfO113 gene performed earlier revealed that this putative virulence marker was present in all porcine isolates, only in 5 strains of bovine origin but in none of E. coli O157 recovered from humans. All 90 E. coli O157 strains tested displayed 10 biochemical profiles that were different at least in one of the reaction tested. The most common atypical reaction observed among porcine O157 isolates was ability to ferment sorbitol (all strains) and production of β-glucuronidase (25 isolates). Moreover, none of the sorbitol-positive strains was able to produce indol. Four antimicrobial resistance profiles among 90 E. coli O157 strains tested were observed. Most of the isolates recovered from humans and all strains from cattle were resistant only to rifampicin whereas the porcine strains showed resistance to either 3 antimicrobials (4 isolates) or to 4 drugs tested (34 isolates). The phenotypic data shown in the present study, together with the previously published genotypic analyses of these strains, confirm earlier suggestions that the porcine E. coli O157 strains are mostly different from those of bovine and human O157 isolates and could therefore play less important role in human STEC O157 infections.