Recombinant immunotoxins are fusion proteins composed of Fv regions of antibodies and bacterial or plant toxins that are being developed for the targeted therapy of cancer. MR1(Fv)-PE38 is a single-chain recombinant immunotoxin that targets a mutant form of the epidermal growth factor receptor (EGFR), EGFRvIII, that is frequently overexpressed in malignant glioblastomas. We have used random complementarity determining region (CDR) mutagenesis to obtain mutants of MR1(Fv) with an increased affinity for EGFRvIII and an increased activity when converted to a recombinant immunotoxin. Initially, nine residues of heavy chain CDR3 were randomly mutagenized, and several mutants with increased binding affinity were isolated. All mutations were located at amino acids 98 and 99, which correspond to a DNA hot spot, a DNA sequence that mutates at high frequency during natural antibody maturation. A specific region of variable region of antibody light chain CDR3 was mutagenized that corresponded to a hot spot and a mutant (MR1-1) with an additional increase in affinity, and cytotoxic activity was isolated. These studies show that targeting hot spots in the CDRs of Fvs is an effective approach to obtaining Fvs with increased affinity. The increased affinity of MR1-1(Fv) makes it an attractive candidate for the targeted therapy of glioblastomas.