Many nonhuman proteins have useful pharmacological activities, but are infrequently effective in humans because of their high immunogenicity. A recombinant immunotoxin (HA22, CAT8015, moxetumomab pasudotox) composed of an anti-CD22 antibody variable fragment fused to PE38, a 38-kDa portion of Pseudomonas exotoxin A, has produced many complete remissions in drug-resistant hairy-cell leukemia when several cycles of the agent can be given, but has much less activity when antibodies develop. We have pursued a strategy to deimmunize recombinant immunotoxins by identifying and removing B-cell epitopes. We previously reported that we could eliminate most B-cell epitopes using a combination of point mutations and deletions. Here we show the location and amino acid composition of all of the B-cell epitopes in the remaining 25-kDa portion of Pseudomonas exotoxin. Using this information, we eliminated these epitopes to produce an immunotoxin (HA22-LR-8M) that is fully cytotoxic against malignant B-cell lines, has high cytotoxic activity against cells directly isolated from patients with chronic lymphocytic leukemia, and has excellent antitumor activity in mice. HA22-LR-8M does not induce antibody formation in mice when given repeatedly by intravenous injection and does not induce a secondary antibody response when given to mice previously exposed to HA22. HA22-LR-8M also has greatly reduced antigenicity when exposed to sera from patients who have produced antibodies to HA22. The properties of HA22-LR-8M make it an excellent candidate for further clinical development.