Neurofilaments purified from invertebrate giant axons have been analyzed with the electron microscope. The neurofilaments have a helical substructure which is most easily observed when the neurofilaments are partially denatured with 0.5 M KCl or 2 M urea. When the ropelike structure comprising the neurofilaments untwists, two strands 4--5.5nm in diameter can be resolved. Upon further denaturation these strands break up into rod-shaped segments and subsequently these segments roll up into amorphous globular structures. Stained, filled densities can be resolved within the strand segments, and these resemble similar structures observed within the intact neurofilaments. The strands appear to consist of protofilaments 2--2.5 nm in diameter. These observations suggest that the neurofilament is a ropelike, helical structure composed of two strands twisted tightly around each other, and they su-port the filamentous rather than the golbular model of intermediate filament structure.