Thirty-two clinical isolates of Shiga-like toxin (SLT)-producing Escherichia coli associated with single cases or outbreaks of bloody diarrhea, hemorrhagic colitis, the hemolytic uremic syndrome, or edema disease of swine were examined for multiple copies of genes belonging to the slt-I or slt-II toxin families. Five of 19 strains that were known to produce SLT-II or to hybridize to slt-II-specific probes by colony blot were found by Southern hybridization to contain two copies of toxin genes related to slt-II. The genes for two toxins closely related to slt-II were cloned from one of the isolates, Escherichia coli O157:H- strain E32511. One copy of the operon was found to be essentially identical to slt-II; it differed from slt-II by only one nucleotide base. This single nucleotide difference did not affect the predicted amino acid sequence. The predicted amino acid sequence of the A subunit of the second operon was identical to that of SLT-II, but the predicted amino acid sequence of the B subunit was identical to that of the B2F1 toxin VT2ha. We designated this second operon slt-IIc. Neutralization assays using several monoclonal antibodies and polyclonal antiserum prepared against SLT-II showed that SLT-IIc was antigenically related to but distinct from SLT-II.