The basement membrane of human corneal epithelial cells (HCECs) has a three-dimensional nanoscale architecture, which includes pores, bumps and fibers that may influence cell–substrate adhesion and spreading in the overlying cells. We previously demonstrated that nano- and microscale groove and ridge patterns influence the morphological response and the adhesive response of HCECs to a nominal wall shear stress. Cell–substrate adhesion is mediated by adhesion receptors that bind to extracellular matrix components and anchor the cytoskeleton (CSK) of cells to extracellular elements. Here we investigate the CSK organization in SV40-transformed HCECs grown on nano- and microscale groove and ridge patterns. X-ray lithography was used to fabricate uniform groove and ridge patterns with features ranging in size from 200 nm to 2 µm grooves. Scanning electron microscopy and transmission electron microscopy were used to investigate CSK structure and the distribution of -β1 integrin adhesion receptors. CSK elements aligned with the patterns; however, the spatial organization of these elements was influenced by feature size. Larger CSK bundles lay on top of the ridges and ran parallel to the patterns, whereas smaller CSK bundles, whose width was proportional to the groove size, spanned the grooves. -β1 integrins co-localized with the CSK and had a higher density at the poles of aligned spindle-shaped cells. Differences in organization seen on the different topographical feature sizes may be indicative of differences in extracellular matrix organization. This may explain, in part, previous observations regarding the dependence of cell adhesive responses on the size of topographic features in the substrate. SCANNING 30: 405–413, 2008. © 2008 Wiley Periodicals, Inc.