Diacetyl (DA), an ubiquitous butter-flavoring agent, was found to influence several aspects of amyloid-β (Aβ) aggregation-one of the two primary pathologies associated with Alzheimer's disease. Thioflavin T fluorescence and circular dichroism spectroscopic measurements revealed that DA accelerates Aβ1-42 aggregation into soluble and ultimately insoluble β-pleated sheet structures. DA was found to covalently bind to Arg 5 of Aβ1-42 through proteolytic digestion-mass spectrometric experiments. These biophysical and chemical effects translated into the potentiation of Aβ1-42 cytotoxicity by DA toward SH-SY5Y cells in culture. DA easily traversed through a MDR1-MDCK cell monolayer, an in vitro model of the blood-brain barrier. Additionally, DA was found not only to be resistant to but also inhibitory toward glyoxalase I, the primary initiator of detoxification of amyloid-promoting reactive dicarbonyl species that are generated naturally in large amounts by neuronal tissue. In light of the chronic exposure of industry workers to DA, this study raises the troubling possibility of long-term neurological toxicity mediated by DA.