In contrast to the general tendency of hydrophobicity-toxicity relationship of amyloid β peptide, we have previously found that the replacement of Asn27 of amyloid β(25–35) peptide with Ala yielded a more hydrophobic but less toxic analog and that of Met35 gave a less hydrophobic but more toxic one. To reveal the unique role of these two residues in the neurotoxicity of amyloid β(1–42) peptide, the major peptide constituent of amyloid plaques in human brain, we synthesized two analogs N27A and M35A in which Asn27 and Met35 of amyloid β(1–42) peptide was replaced with Ala, respectively. The former showed much weaker toxicity than the native peptide, while the latter showed almost an equivalent toxicity, indicating that the side chain amide group of Asn27 has an essential role for the toxicity of amyloid β peptides.