Amyloid fibrils are assemblies of misfolded proteins and are associated with pathological conditions such as Alzheimer's disease and the spongiform encephalopathies. In the amyloid diseases, a diverse group of normally soluble proteins self‐assemble to form insoluble fibrils. X‐ray fibre diffraction studies have shown that the protofilament cores of fibrils formed from the various proteins all contain a cross‐β‐scaffold, with β‐strands perpendicular and β‐sheets parallel to the fibre axis. We have determined the threedimensional structure of an amyloid fibril, formed by the SH3 domain of phosphatidylinositol‐3′‐kinase, using cryo‐electron microscopy and image processing at 25 Å resolution. The structure is a double helix of two protofilament pairs wound around a hollow core, with a helical crossover repeat of∼ 600 Å and an axial subunit repeat of∼ 27 Å. The native SH3 domain is too compact to fit into the fibril density, and must unfold to adopt a longer, thinner shape in the amyloid form. The 20× 40‐Å protofilaments can only accommodate one pair of flat β‐sheets stacked against each other, with very little inter‐strand twist. We propose a model for the polypeptide packing as a basis for understanding the structure of amyloid fibrils in general.