The iota toxin of Clostridium perfringens type E is a guinea pig dermonecrotic, mouse lethal toxin which cross-reacts with the iota-like toxin of Clostridium spiroforme. Antiserum raised against C. spiroforme or C. perfringens type E neutralizes the toxin from both species. By using C. spiroforme antiserum and crossed immunoelectrophoresis, we have found that there are two cross-reacting proteins, designated iota a (ia) and iota b (ib) in the culture filtrate of C. perfringens type E. Both proteins of C. perfringens were separated by preparative isoelectric focusing and had very little toxic activity when tested alone. However, when they were recombined there were 8- and 25-fold increases in bioactivity as determined by mouse lethal and guinea pig dermonecrotic assays, respectively. These results demonstrate that the iota toxin of C. perfringens requires two immunologically and biochemically different proteins for maximum activity.