Recombinant immunotoxins, containing an Fv fragment and a bacterial toxin, frequently elicit neutralizing antibodies, nearly always against the toxin. Moxetumomab pasudotox (previously called CAT-8015 or HA22) contains an anti-CD22 Fv fused to PE38, a truncated form of Pseudomonas exotoxin, containing amino acids 253-364 and 381-613. One avenue to reducing immunogenicity is to identify B- and T-cell epitopes and remove them while retaining toxin activity. To determine B-cell epitopes on PE38, 60 monoclonal antibodies against PE38 were tested in a pairwise manner, and seven major epitope groups with 13 subgroups were identified. The locations of many of these epitopes were identified by mutating large surface-exposed residues to alanine. A mutant of moxetumomab pasudotox containing eight epitope-eliminating mutations (HA22-8X) was prepared, and greatly reduced immunogenicity in mice. In parallel, two large sections of PE38 containing lysosomal protease cleavage sites were removed, leaving only amino acids 274-284 and 394-613 of the toxin. The resulting molecule, HA22-LR, retained cytotoxicity toward CD22+ cell lines, killed primary chronic lymphocytic leukemia cells more potently than moxetumomab pasudotox, was much less toxic to mice, and had significantly improved antitumor activity toward murine xenografts. The immunogenicity and activity of recombinant immunotoxins may be optimized by combinations of these approaches.