EGFRvIII is a mutant epidermal growth factor receptor found in glioblastoma, and in carcinoma of the breast, ovary, and lung. The mutant receptor has a deletion in its extracellular domain that results in the formation of a new, tumor-specific extracellular sequence. Mice were immunized with a synthetic peptide corresponding to this sequence and purified EGFRvIII. A single chain antibody variable domain (scFv) phage display library of 8 ؋ 10 6 members was made from the spleen of one immunized mouse. A scFv specific for EGFRvIII was isolated from this library by panning with successively decreasing amounts of synthetic peptide. This was used to make an immunotoxin by fusing the scFv DNA sequence to sequences coding for domains II and III of Pseudomonas exotoxin A. Purified immunotoxin had a K d of 22 nM for peptide and a K d of 11 nM for cell-surface EGFRvIII. The immunotoxin was very cytotoxic to cells expressing EGFRvIII, with an IC 50 of 1 ng͞ml (16 pM) on mouse fibroblasts transfected with EGFRvIII and an IC 50 of 7-10 ng͞ml (110-160 pM) on transfected glioblastoma cells. There was no cytotoxic activity at 1000 ng͞ml on the untransfected parent glioblastoma cell line. The immunotoxin was completely stable upon incubation at 37؇C for 24 h in human serum. The combination of good affinity, cytotoxicity and stability make this immunotoxin a candidate for further preclinical evaluation.Immunotoxins are therapeutic agents for cancer that consist of a targeting molecule linked to a cytotoxic agent (1). Antibodies, or genetically engineered antibody variable domains (Fvs), are usually used for targeting. Our laboratory has focused on using the protein Pseudomonas exotoxin A as a cytotoxic agent. Pseudomonas exotoxin A is a three-domain protein (2): domain I binds to the ␣2 macroglobulin receptor, which internalizes the toxin (3); domain II mediates translocation of the toxin to the cell cytosol; domain III ADP ribosylates elongation factor 2 leading to arrest of protein synthesis and cell death. The toxin can be converted to a cancer therapeutic by replacing domain I with binding domains that are selective for cancer cells. Recently our laboratory completed a phase I trial of an immunotoxin made with an antibody attached to domains II and III of Pseudomonas exotoxin A (4). The antibody was specific for a Lewis Y-related carbohydrate antigen that is overexpressed in many cancers. Tumor regressions were seen in several patients with advanced breast and colorectal cancer, demonstrating that immunotoxins made with Pseudomonas exotoxin A do have activity against solid tumors in humans.