Ankyrin_G 480/270 kDa and three ankyrin-binding integral membrane proteins (neurofascin, NrCAM, and the voltage-dependent sodium channel) colocalize within a specialized domain of the spectrin–actin network found at axonal segments of nodes of Ranvier in myelinated axons. Before myelination in embryonic nerves, ankyrin_G480/270 kDa and the related ankyrin isoform ankyrin_B 440 kDa are co-expressed along with NrCAM in an abundant, continuous distribution along the length of axons. This study has resolved intermediate stages in the developmental transition from a continuous distribution of ankyrin_G 480/270 kDa in all axons to a highly polarized localization at the node of Ranvier in the developing rat sciatic nerve. The first detected event is formation of clusters containing the cell adhesion molecules neurofascin and NrCAM at sites independent of myelin-associated glycoprotein (MAG)-staining Schwann cell processes. Subsequent steps involve recruitment of ankyrin_G 480/270 kDa and the voltage-dependent sodium channel to cluster sites containing cell adhesion molecules, and elaboration of MAG-staining Schwann cell processes adjacent to these cluster sites. Formation of the mature node of Ranvier results from the fusion of asynchronously formed pairs of clusters associated with MAG-positive Schwann cells flanking the site of presumed node formation. Studies with the hypomyelinating mutant mousetrembler demonstrate that the elaboration of compact myelin is not required for the formation of these clustered nodal intermediates. Clustering of neurofascin and NrCAM precedes redistribution of ankyrin_G 480/270 kDa and the voltage-dependent sodium channel, suggesting that the adhesion molecules define the initial site for subsequent assembly of ankyrin and the voltage-dependent sodium channel.