Growth of cells in tissue culture is generally performed on two-dimensional (2D) surfaces composed of polystyrene or glass. Recent work, however, has shown that such 2D cultures are incomplete and do not adequately represent the physical characteristics of native extracellular matrix (ECM)/basement membrane (BM), namely dimensionality, compliance, fibrillarity, and porosity. In the current study, a three-dimensional (3D) nanofibrillar surface composed of electrospun polyamide nanofibers was utilized to mimic the topology and physical structure of ECM/BM. Additional chemical cues were incorporated into the nanofibrillar matrix by coating the surfaces with fibronectin, collagen I, or laminin-1. Results from the current study show an enhanced response of primary mouse embryonic fibroblasts (MEFs) to culture on nanofibrillar surfaces with more dramatic changes in cell spreading and reorganization of the cytoskeleton than previously observed for established cell lines. In addition, the cells cultured on nanofibrillar and 2D surfaces exhibited differential responses to the specific ECM/BM coatings. The localization and activity of myosin II-B for MEFs cultured on nanofibers was also compared. A dynamic redistribution of myosin II-B was observed within membrane protrusions. This was previously described for cells associated with nanofibers composed of collagen I but not for cells attached to 2D surfaces coated with monomeric collagen. These results provide further evidence that nanofibrillar surfaces offer a significantly different environment for cells than 2D substrates.