We have previously demonstrated that Escherichia coli DH5 alpha clones expressing closely-related Shiga-like toxin type II operons (designated SLT-II/OX3b and SLT-II/O48) had similar cytotoxicity for Vero cells, but differed in oral virulence for streptomycin-treated mice. Studies with chimeric toxin operons indicated that increased virulence was associated with the A subunit of SLT-II/OX3b, which differs from that of SLT-II/O48 by two amino acids (at positions 4 and 102). In the present study, we have constructed a series of additional chimeric derivatives of the SLT-II/OX3b and SLT-II/O48 operons and assessed the effect of single A subunit amino acid substitutions on oral virulence. Maximal virulence, as judged by median survival time after oral challenge, was associated only with the combination of Met4 and Gly102, as found in the A subunit of SLT-II/OX3b.