Clostridium perfringens enterotoxin (CPE), a single polypeptide of 319 amino acids, has a unique multistep mechanism of action. In the first step, CPE binds to claudin proteins and/or a 50-kDa eukaryotic membrane protein receptor, forming a small (~90-kDa) complex. This small complex apparently then associates with a 70-kDa eukaryotic membrane protein, resulting in formation of a large complex that induces the onset of membrane permeability alterations. To better define the boundaries of CPE functional regions and to identify specific amino acid residues involved in various steps of CPE action, in this study we subjected the cloned cpe gene to random mutagenesis in XL-1 Red strains of Escherichia coli. Seven CPE random mutants with reduced cytotoxicity for Vero cells were phenotypically characterized for the ability to complete each step in CPE action. Five of these seven recombinant CPE (rCPE) random mutants (G49D, S59L, R116S, R137G, and S167P) exhibited binding characteristics similar to those of rCPE or native CPE, while the Y310C and W226Stop mutants showed reduced binding and no binding, respectively, to brush border membranes. Interestingly, two completely nontoxic mutants (G49D and S59L) were able to bind and form small complex but they did not mediate any detectable large complex formation. Another strongly attenuated mutant, R116S, formed reduced amounts of an anomalously migrating large complex. Collectively, these results provide further support for large complex formation being an essential step in CPE action and also identify the CPE region ranging from residues ~45 to 116 as important for large complex formation. Finally, we also report that limited removal of extreme N-terminal CPE sequences, which may occur in vivo during disease, stimulates cytotoxic activity by enhancing large complex formation.