Tissue development in multicellular animals relies on the ability of cells to synthesize an extracellular matrix (ECM) containing spatially organized collagen fibrils, whose length greatly exceeds that of individual cells. The importance of the correct regulation of fibril deposition is exemplified in diseases such as osteogenesis imperfecta (caused by mutations in collagen genes), fibrosis (caused by ectopic accumulation of collagen), and cardiovascular disease (which involves cells and macromolecules binding to collagen in the vessel wall). Much is known about the molecular biology of collagens but less is known about collagen fibril structure and how the fibrils are formed (fibrillogenesis). This is explained in part by the fact that the fibrils are noncrystalline, extensively cross-linked, and very large, which makes them refractory to study by conventional biochemical and high-resolution structure-determination techniques. Electron microscopy has become established as the method of choice for studying collagen fibril structure and assembly. This article describes the electron microscopic methods most often used in studying collagen fibril assembly and structure. © 2008 Elsevier Inc. All rights reserved.