Amiloride-sensitive sodium channels are localized to the microvillar domain of apical membranes in sodium-transporting renal epithelial cells. To elucidate the elements that maintain sodium channel distribution at the apical membrane, we searched for specific proteins associating with the channel. Triton X-100 extraction of A6 epithelial cells reveals that sodium channels are associated with detergentinsoluble and assembled cytoskeleton. Indirect immunofluorescence and confocal microscopy show that sodium channels are segregated to the apical microvillar membrane and colocalize with ankyrin, fodrin, and actin. We document by immunoblot analysis that ankyrin and fodrin remain associated with sodium channels after isolation and purification from bovine renal papillae. 125I-labeled ankyrin can be precipitated by anti-sodium-channel antibodies only in the presence of purified bovine sodium-channel complex. Direct binding of 125I-labeled ankyrin shows ankyrin binds to the 150-kDa subunit of the channel. Fluorescence photobleach lateral-diffusion measurements indicate sodium channels are severely restricted in their lateral mobility. We conclude that ankyrin links the amiloride-sensitive sodium channel to the underlying cytoskeleton and this association may sequester sodium channels at apical microvilli and maintain their polarized distribution in renal epithelial cells. (.