Clostridium perfringens type E iota toxin is composed of two separate and independent polypeptide chains that act synergistically in mouse lethal assays. The light chain is an enzyme that mono(ADP-ribosyl)ates certain amino acids. The enzyme displays substantial activity when homopoly-L-arginine is used as a substrate, but it shows little activity when polyasparagine, polylysine or polyglutamic acid are used. In keeping with the properties of an ADP-ribosylating enzyme, the toxin possesses the following characteristics. It produces incorporation of radioactivity into polyarginine when adenine-labeled NAD is used, but radioactivity is not incorporated when nicotinamide-labeled NAD is used. Irrespective of labeling, enzymatic activity is accompanied by the release of free nicotinamide. After incorporation of ADP-ribose groups into polyarginine, enzymatic and chemical techniques can be used to release the incorporated material. Snake venom phosphodiesterase releases mainly AMP; hydroxylamine releases AMP and ADP-ribose. The heavy chain of iota toxin has little or no enzyme activity, and it does not substantially affect the enzyme activity of the light chain. The heavy chain may be a binding component that directs the toxin to vulnerable cells. The data suggest that iota toxin is a representative of a novel class of ADP-ribosylating toxins.