Glucagon solutions at pH 2.0 were subjected to mechanical agitation at 37 degrees C in the presence of a hydrophobic surface to explore the details of aggregation and fiber formation. High-resolution intermittent-contact atomic force microscopy performed in solution revealed the presence of aggregates after 0.5 h; however, longer agitation times resulted in the formation of fibrillated structures with varying levels of higher-order assembly. Height, periodicity, and amplitude measurements of these structures allowed the identification of four distinct fiber types. The most elementary fiber form, designated a filament, self-associates in a specific wound fashion to produce protofibrils composed of two filaments. Subsequent self-assembly of these filaments and protofibrils leads to two well-defined fibrillar motifs, termed Type I and Type II. Atomic force microscopy imaging of pH 2.8 glucagon solutions not agitated or exposed to elevated temperature revealed the presence of amorphous aggregates before the formation of fibrillar structures similar to those seen at pH 2.0. Time-course solution Fourier transform infrared spectroscopy and thioflavin T binding studies suggested that glucagon aggregation and fibril formation were associated with the development of beta-sheet structure. The results of these studies are used to describe a possible mechanism for glucagon aggregation and fibrillation that is consistent with a hierarchical assembly model proposed for amyloid fibril formation.