&Toxin (perfringolysin 0), secreted by Clostridium perfringens, shares with other known thiol-activated toxins a conserved undecapeptide, ECTGLAWEWWR, located in the C-terminal region of the protein and containing the unique cysteine of the molecule. Single and double amino acid substitutions were created in the 8-toxin molecule to investigate the role of individual tryptophan residues in the lytic activity of &toxin. Wild-type and mutant &toxins were overproduced in Escherichia coli by means of a T7 RNA polymerase/promoter system and purified. The relative hemolytic activities of four mutant toxins, each with a Trp to Phe substitution outside the common Cys-containing region, were more than 60% that of wild-type 8-toxin. In contrast, mutant toxins with Phe replacements within the Cys-containing region (at Trp436, Trp438 or Trp439) showed significantly reduced hemolytic and erythrocyte-membranebinding activities. The largest reduction in binding affinity, more than 100-fold, was observed for Trp438 mutant toxins. However, the mutants retain binding specificity for cholesterol and the ability to form arcshaped and ring-shaped structures on membranes. These results indicate that the low hemolytic activities of these mutant toxins can be ascribed, at least in part, to reduced binding activities. With respect to protease susceptibility and far-ultraviolet circular-dichroism spectra, only the W436-F mutant toxin, showed any considerable difference from wild-type toxin in secondary or higher-order structures, indicating that Trp436 is essential for maintenance of toxin structure.