Studies to define the mechanisms by which the extracellular matrix (ECM) activates Rho GTPases within the cell have generally focused on the chemistry of the macromolecules comprising the ECM. Considerably less information is available to assess the role of the physical structure of the ECM, particularly its three dimensional (3D) geometry. In this report, we examined the effect of 3D surfaces on the activation states of Rho GTPases within NIH 3T3 fibroblasts and normal rat kidney cells. Cells were cultured on synthetic 3D surfaces comprised of polyamide nanofibers. In contrast to results using two dimensional tissue culture surfaces, growth of both cell types on 3D nanofibrillar surfaces resulted in a preferential and sustained activation of the small GTPase Rac. These results support the growing view that in addition to chemical composition, the three dimensionality and nanofibrillar architecture of ECM may represent another essential element in signal transduction pathways and cellular physiology.