The peptide hormone insulin forms a spherical aggregate, called a spherulite, at low pH and high temperature. A spherulite is composed of a core and many fibrils extending from it. These fibrils are thought to be amyloid fibers with a beta-sheet structure. In the present study, spherulites with a diameter of 50-100 microm were examined by X-ray fiber diffraction using a 6 microm beam. When a spherulite was scanned with the microbeam and the observed diffraction patterns were arranged in a two-dimensional array, the direction of the scatter was centrosymmetric, demonstrating a symmetric growth of fibrils. There were diffraction peaks at Bragg spacings of 23 nm, 3.3 nm and 1.2 nm in the direction perpendicular to the fibrils and 0.48 nm along the fibrils. The 0.48 nm reflection shows that the hydrogen bonds between beta-strands are along the fibril. The 23 nm reflection corresponds to the separation between fibrils, the 3.3 nm reflection is due to the arrangement of protofilaments, and the 1.2 nm reflection arises from the arrangement of peptide chains. On the basis of these results, a model of a fibril with an extended insulin molecule is proposed.