The purpose of this study was to assess a simplified method for interstrain differentiation of Escherichia coli O157:H7 and other Shiga-like toxin-producing E. coli (SLTEC) strains. A method based on the use of nucleic acid probes from Shiga-like toxin (SLT) I and II structural genes was used to generate restriction fragment length polymorphism (RFLP) patterns of SLTEC strains, (SLT-RFLP patterns) resulting from digestion of isolated genomic DNA with four different restriction enzymes (BamHI, EcoRI, HindIII, and PvuII) used separately. A total of 165 SLTEC strains from clinical, food, and environmental sources, including O157:H7 isolates from four food-borne outbreaks in Canada and the United States, were analyzed in the study. SLT-RFLP demonstrated that E. coli O157:H7 strains from each food-borne outbreak had the same unique SLT-RFLP pattern. Fifty-two SLT-RFLP types were found among 96 E. coli O157:H7 isolates from sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome in Washington state. The use of the SLT probes proved to be a very powerful method for interstrain differentiation of SLTEC strains. Although the use of each of the enzymes alone did not give enough differentiative power to be used in epidemiological studies, the combination of patterns generated by two restriction enzymes (EcoRI and PvuII, used separately) provided the desired sensitivity for such studies. The results clearly demonstrate the usefulness of the method for studying the molecular epidemiology of E. coli O157:H7. The method is also suitable for establishing an epidemiological database, in terms of both sensitivity and ease of compilation and interpretation of results.