Accumulation of amyloid beta-peptide (Abeta) and tau aggregates, possibly linked to age-associated deficiencies in protein homeostasis, appear to cause Alzheimer's disease. Schiff-base formation between Abeta and the aldehyde-bearing cholesterol oxidation product 3-beta-hydroxy-5-oxo-5,6-secocholestan-6-al is known to increase Abeta amyloidogenicity. Here, we synthesized Abeta variants site-specifically modified with the cholesterol aldehyde at Asp-1, Lys-16, or Lys-28, rather than studying mixtures. These distinct modifications have a similar effect on the thermodynamic propensity for aggregation, enabling aggregation at low concentrations. In contrast, the modification site differentially influences the aggregation kinetics; Lys-16-modified Abeta formed amorphous aggregates fastest and at the lowest concentration (within 2 h at a concentration of 20 nM), followed by the Lys-28 and Asp-1 conjugates. Also, the aggregates resulting from Abeta Lys-16 cholesterol aldehyde conjugation were more toxic to primary rat cortical neurons than treatment with unmodified Abeta under identical conditions and at the same concentration. Our results show that Abeta modification by cholesterol derivatives, especially at Lys-16, renders it kinetically and thermodynamically competent to form neurotoxic aggregates at concentrations approaching the physiologic concentration of Abeta.