Thin films of the extracellular matrix protein, collagen, were prepared by adsorbing native or heat-denatured type I collagen onto hexadecanethiol self-assembled monolayers. The resulting films were characterized by atomic force microscopy, ellipsometry, and light microscopy. Denatured collagen formed a topographically smooth similar to3.6 nm thick film, consistent with an adsorbed protein monolayer. In contrast, the native collagen thin film consisted of supramolecular collagen fibrils. The density of the large fibrils could be varied by changing the native collagen concentration in the solution from which the films were prepared. The biomimetic nature of the thin collagen films was partially assessed by examining their effects on vascular smooth muscle cells. Automated quantitative analysis indicated that the morphology of smooth muscle cells on the thin films was dependent on whether the collagen was heat-denatured or was in its native fibrillar form. The area of cells on denatured collagen films was significantly larger than that of cells on thin films of native fibrillar collagen. This response closely mimicked the response of these cells to thick collagen gels. Examination of the relationship between collagen fibril density and cell area indicated that large fibrils play a role in determining how cells respond to collagen. Cells assumed a larger morphology on native collagen films with a lower density of large fibrils. In this study, it is clear that cell morphology on these films is determined by micron-scale interactions between cells and the matrix molecules and is not dependent on the bulk materials properties of collagen gels.