We used recombinant DNA technology to construct a mutant form of Pseudomonas exotoxin A (PE) called cysPE35 that contains amino acids 280-364 and 381-613 of PE. cysPE35 begins at the native PE proteolytic cleavage site and contains a single cysteine residue at position 287 that can be used to conjugate the toxin to monoclonal antibodies (MAbs). Unlike immunotoxins containing larger mutant forms of PE, such as PE40 or PE38, immunotoxins containing cysPE35 linked through a disulfide bond do not require proteolysis to generate a toxin fragment able to translocate to the cytosol. cysPE35 was conjugated to several MAbs and their activities were studied in vitro and in vivo. The concentration of toxin that inhibited protein synthesis as measured by a decrease in [3H]leucine incorporation of 50% of cysPE35 conjugated through a disulfide bond to the MAb HB21, which targets the human transferrin receptor, was 1 ng/ml on A431 cells. The MAb HB21 conjugated through a thioether bond to cysPE35 was much less active (concentration of toxin that inhibited protein synthesis as measured by a decrease in [3H]leucine incorporation of 50%, 200 ng/ml). An immunotoxin containing PE38 conjugated through either a disulfide or thioether bond to the MAb HB21 had a concentration of toxin that inhibited protein synthesis as measured by a decrease in [3H]leucine incorporation of 50% of 5 ng/ml, indicating that proteolysis of PE38 may be rate limiting in the action of these immunotoxins. Two other MAbs, LL2 and B3, were also conjugated through a disulfide bond to cysPE35. Both immunotoxins were also more active against cultured cells than conjugates using PE38 or PE40, and caused complete regression of human tumor xenografts growing in nude mice. In conclusion, we have constructed a mutant form of PE which must be coupled to MAbs through a disulfide bond to produce fully active immunotoxins that do not require proteolysis to generate a toxin fragment able to reach the cell cytosol.