The maximal number of norleucine methyl ester (NME) incorporated into carboxyl groups in epsilon toxin of Clostridium perfringens by the carbodiimide-nucleophile procedure was 7 and 17 in the absence and presence of 8 M urea, respectively. The introduction of 3-4 nucleophilic modifying agents such as NME, glycine methyl ester or taurine into carboxyl groups of the toxin reduced the lethality to approximately 10% of the original activity. The incorporation of 6-7 of these agents resulted in complete loss of the activity. On the other hand, circular dichroism spectra and the reaction between the toxin or the NME-incorporated toxin and antiepsilon toxin reaction showed no difference between the intact toxin and the NME-incorporated toxin. The data suggested that at least 4 out of 7 carboxyl groups on the surface of the toxin are important in maintaining the lethal activity of toxin.