Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and electron microscopy (EM) have been used simultaneously to follow the temperature-induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native-like α-helical structure. On heating to 70 °C, partial unfolding occurs and results initially in aggregates that are shown by CD and FT-IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the molecular conformation from α-helical to β-sheet structure. At later stages, EM shows the development of fibrils with well-defined repetitive morphologies including structures with a periodic helical twist of ∼450 Å. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphology of the initially formed fibrillar species.