Structural studies on biological macromolecules, both monomeric and as organized into higher order assemblies, have been performed by high resolution electron microscopy complemented with optical diffraction and computer image processing. Intermediate filaments (IF) are important constituents of the cytoskeletons of virtually every type of eukaryotic cell, but structural information on IF has remained scanty. We have analyzed digital micrographs of several types of IF imaged by dark-field scanning transmission electron microscopy (STEM) without recourse to contrast enhancement by heavy metal staining or shadowing. (i) By comparing native vimentin IF with filaments reassembled from purified subunits, we have performed a rigorous test of the authenticity of this in vitro assembly product. (ii) Taken together, our mass-per-unit-length measurements on several keratin IF types indicate that their linear densities are proportional to the average molecular weights of the constituent subunits. Other data indicate that this homology also extends to vimentin and desmin IF. Its basis appears to derive from a common structural building-block similarly polymerized in all these cases, an interpretation that accords well with emerging amino-acid sequence data on IF subunits. (iii) We find that the mass density of IF extends to an outermost radial limit of 7-8 nm in a diffuse peripheral shell surrounding a 4-5 nm radius core of approximately uniform density. These findings indicate that earlier measurements of IF diameter (8-10 nm) by traditional electron microscopic methods are likely to be substantial underestimates. Further structural studies have been performed on the radial organization of Vesticular Stomatitis Virus, a representative rhabdovirus, by computer analysis of CTEM images of specifically stained thin sections and of STEM micrographs; on clathrin-coated vesicles purified from bovine brain and rat liver, with particular attention to the polymorphism encountered amoung these particles; and on protein paracrystals formed out of human psoriatic keratin.