Dragline fibers collected at a controlled silking rate from Nephila clavipes spiders were studied by analytical transmission electron microscopy (TEM). The physical microstructure consists of irregularly-shaped ß-sheet crystallites (approx. 70–100 nm in diameter, and comprising approx. 50 volume per cent) in an amorphous matrix. Electron diffraction from single crystallites indicates an orthogonal unit cell with space group P21; the lattice parameters are a = 13.31 Å (inter-sheet repeat), b = 9.44 Å (inter-chain repeat within ß-sheets) and c = 20.88 Å (intra-chain repeat). The crystallites are not truly periodic structures having long range positional order, but are better described as non-periodic layered structures, similar to the ordered structures formed when liquid crystalline random copolymers solidify. Electron energy loss spectroscopy (EELS) reveals chemical microstructure in the fibers. The matrix contains bands (approx. 200 nm wide) of alternately higher and lower nitrogen content. This variation cannot be explained in terms of known protein domains alone, and may reflect the presence of retained small-molecule species from secretory granules. Calcium is localized uniquely to the crystallites (at a concentration of approx. one calcium atom per 120 amino acid residues, as measured by inductively coupled plasma spectroscopy). Varying the calcium content may be one mechanism by which the spider can tailor the evolution of the microstructure, and hence the physical properties of the silk fiber.