EGFRvIII is a cancer-specific epidermal growth factor tyrosine kinase receptor mutation, expressed in different kinds of cancer, in particular ovarian, glioblastomas, and breast cancer. A peptide, PEPHC1, has previously been shown to bind selectively to EGFRvIII. An alanine scan was performed to identify the amino acid residues important for binding of PEPHC1 to EGFRvIII. The results indicate that the amino acid residues at the N-terminus of PEPHC1 are essential for the binding to the mutated receptor. One analog, [Ala(12)]PEPHC1, showed higher selective binding to EGFRvIII than PEPHC1. On the basis of these results, six truncated peptide analogs derived from the N-terminus of PEPHC1, H-HFIIL-NH2, H-HFIILG-NH2, H-HFIILGF-NH2, H-HFIILGFM-NH2, H-HFLIIGFMR-NH2, and H-HFLIIGFMRR-NH2 were synthesized and tested in the same manner. We observed that H-HFIIL-NH2 and H-HFIILG-NH2 showed almost threefold lower binding to the mutated receptor than PEPHC1, whereas the remainder showed 25% lower binding. The secondary structure of the PEPHC1 analogs was investigated by far UV circular dichroism spectroscopy and their binding correlated with various structural parameters such as charge, mean hydrophobicity (<H>), and mean hydrophobic moment (<mu(H)>). This work provides data, which will be useful in the development of novel peptide-based ligands for EGFRvIII-targeted diagnostics and therapy. This work was in part presented at the 17th International Symposium on Radiopharmaceutical Sciences, April 2007, Aachen, Germany.