We use two-dimensional IR (2D IR) spectroscopy to explore fibril formation for the two predominant isoforms of the beta-amyloid (A beta(1-40) and A beta(1-42)) protein associated with Alzheimer's disease. Two-dimensional IR spectra resolve a transition at 1610 cm(-1) in A beta fibrils that does not appear in other A beta aggregates, even those with predominantly beta-sheet-structure-like oligomers. This transition is not resolved in linear IR spectroscopy because it lies under the broad band centered at 1625 cm(-1), which is the traditional infrared signature for amyloid fibrils. The feature is prominent in 2D IR spectra because 2D lineshapes are narrower and scale nonlinearly with transition dipole strengths. Transmission electron microscopy measurements demonstrate that the 1610 cm(-1) band is a positive identification of amyloid fibrils. Sodium dodecyl sulfate micelles that solubilize and disaggregate preaggregated A beta samples deplete the 1625 cm(-1) band but do not affect the 1610 cm(-1) band, demonstrating that the 1610 cm(-1) band is due to very stable fibrils. We demonstrate that the 1610 cm(-1) transition arises from amide I modes by mutating out the only side-chain residue that could give rise to this transition, and we explore the potential structural origins of the transition by simulating 2D IR spectra based on A beta crystal structures. It was not previously possible to distinguish stable A beta fibrils from the less stable beta-sheet-rich oligomers with infrared light. This 2D IR signature will be useful for Alzheimer's research on A beta aggregation, fibril formation, and toxicity.