Inhibition of amyloid β-protein (Aβ)-induced toxicity is a promising therapeutic strategy for Alzheimer's disease (AD). Previously, we reported that the C-terminal tetrapeptide Aβ(39-42) is a potent inhibitor of neurotoxicity caused by Aβ42, the form of Aβ most closely associated with AD. Here, initial structure-activity relationship studies identified key structural requirements, including chirality, side-chain structure, and a free N-terminus, which control Aβ(39-42) inhibitory activity. To elucidate the binding site(s) of Aβ(39-42) on Aβ42, we used intrinsic tyrosine (Y) fluorescence and solution-state NMR. The data suggest that Aβ(39-42) binds at several sites, of which the predominant one is located in the N-terminus of Aβ42, in agreement with recent modeling predictions. Thus, despite the small size of Aβ(39-42) and the hydrophobic, aliphatic nature of all four side-chains, the interaction of Aβ(39-42) with Aβ42 is controlled by specific intermolecular contacts requiring a combination of hydrophobic and electrostatic interactions and a particular stereochemistry.