The key pathogenic event in the onset of Alzheimer's disease (AD) is believed to be the aggregation of the β-amyloid (Aβ) peptide into toxic oligomers. Molecules that interfere with this process may therefore act as therapeutic agents for the treatment of AD. N-Methylated peptides (meptides) are a general class of peptide aggregation inhibitors that act by binding to one face of the aggregating peptide but are unable to hydrogen bond on the other face, because of the N-methyl group replacing a backbone NH group. Here, we optimize the structure of meptide inhibitors of Aβ aggregation, starting with the KLVFF sequence that is known to bind to Aβ. We varied the meptide length, N-methylation sites, acetylation, and amidation of the N and C termini, side-chain identity, and chirality, via five compound libraries. Inhibitor activity was tested by thioflavin T binding, affinity chromatography, electron microscopy, and an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide toxicity assay. We found that inhibitors should have all D chirality, have a free N terminus but an amidated C terminus, and have large, branched hydrophobic side chains at positions 1-4, while the side chain at position 5 was less important. A single N-methyl group was necessary and sufficient. The most active compound, D-[(chGly)-(Tyr)-(chGly)-(chGly)-(mLeu)]-NH2, was more active than all previously reported peptide inhibitors. Its related non-N-methylated analogues were insoluble and toxic. © 2006 American Chemical Society.