Neurofilaments in the axons of mammalian spinal cord neurons are extensively cross-linked; consequently, the filaments and their cross-bridges compose a three-dimensional lattice. We have used antibody decoration in situ combined with tissue preparation by the quick-freeze, deep-etch technique to locate three neurofilament polypeptides (195, 145, and 73 Kd) within this lattice. When antibodies against each polypeptide were incubated with detergent-extracted, formaldehyde-fixed samples of rabbit spinal cord, each antibody assumed a characteristic distribution: anti-73-Kd decorated the neurofilament core uniformly, but not the cross-bridges; anti-145-Kd also decorated the core, but less uniformly; sometimes the anti-145-Kd antibodies were located over the bases of cross-bridges. In contrast, anti-195-Kd primarily decorated the cross-bridges between the neurofilaments. These observations show that the 73-Kd polypeptide is a component of the central core of neurofilaments, and that the 195-Kd polypeptide is a component of the inter-neurofilamentous cross-bridges. It is consistent with this conclusion that we found few cross-bridges between neurofilaments in the optic nerves of neonatal rabbits during a developmental period when the ratio of 195 to 73 or 145-Kd polypeptides is much lower than in adults. The ratio of 195-Kd polypeptide to the other two neurofilament polypeptides also appeared much lower in the cell bodies and dendrites than in axons of adult spinal cord neurons, when the dispositions of the three polypeptides were studied by immunofluorescence experiments. The cell bodies apparently contain neurofilaments composed primarily of 145- and 73-Kd polypeptides, because we observed antibody decoration of individual neurofilaments in the cell bodies with anti-73- and -145-Kd, but not with anti-195-Kd. We conclude that the 195-Kd polypeptide participates in a cross-linking function, and that this function is, at least in certain neurons, most prevalent in the mature axon.