To determine the degree of heterogeneity among Shiga-like toxin-II (SLT-II)-related toxins present in enterohemorrhagic Escherichia coli O157 strains, slt-IIB-related genes of 15 strains were amplified and sequenced. Of these 15 isolates, six contained only the slt-II-related genes, seven strains harbored slt-II-related genes together with slt-II, and two strains had slt-II-related genes plus slt-I. In strains carrying slt-II-related genes alone or in combination with slt-I, the PCR fragments were directly subjected to Taq cycle sequence analysis. Direct sequencing was not possible with the seven strains possessing both slt-II and slt-II-related genes, since the PCR products contained both genes. In order to allow sequence analysis of these slt-II-related genes, the PCR products were first subjected to restriction enzyme digestion with FokI, which selectively digested slt-IIB. This resulted in an undigested 270-bp fragment consisting of pure slt-II-related genes. Interestingly, comparison of the nucleotide sequences revealed 100% homology of all analyzed 15 slt-IIB-related toxin genes. In addition, the nucleotide sequence of slt-IIB-related toxin genes were identical to slt-IIcB. Our findings indicate that SLT-IIc is a major variant form of SLT-II present in E. coli O157 strains.