We have utilized electron paramagnetic resonance spectroscopy to study secondary structure, subunit interaction, and molecular orientation of vimentin molecules within intact intermediate filaments and assembly intermediates. Spectroscopy data prove alpha-helical coiled-coil structures at individual amino acids 316-336 located in rod 2B. Analysis of positions 305, 309, and 312 identify this region as conforming to the helical pattern identified within 316-336 and thus demonstrates that, contrary to some previous predictions, this region is in an alpha-helical conformation. We show that by varying the position of the spin label, we can identify both intra- and inter-dimer interactions. With a label attached to the outside of the alpha-helix, we have been able to measure interactions between positions 348 of separate dimers as they align together in intact filaments, identifying the exact point of overlap. By mixing different spin-labeled proteins, we demonstrate that the interaction at position 348 is the result of an anti-parallel arrangement of dimers. This approach provides high resolution structural information (<2 nm resolution), can be used to identify molecular arrangements between subunits in an intact intermediate filament, and should be applicable to other noncrystallizable filamentous systems as well as to the study of protein fibrils.