The insertion of a specific 25-residue internal peptide into ricin toxin A chain (RTA) reduced the catalytic activity of this protein approximately 300-fold. Directed proteolytic cleavage of the peptide insert essentially restored catalytic activity of the resulting two peptide A chain to normal levels. Ricin holotoxin containing unprocessed mutant A chain was not toxic to cultured mammalian cells, due to enhanced proteasomal degradation, nor was it toxic when injected into rats at a concentration that is lethal in the case of native ricin. Rats treated in this way were completely resistant to native ricin when subsequently challenged with a potentially lethal dose of the toxin. These ricin-resistant animals had a significant anti-ricin antibody titer, indicating that this approach has potential for developing an effective vaccine against this toxin.