Recent studies in basic cell biology and bioengineering call for model substrates that present active proteins, with control over protein density, pattern, and orientation, to more directly mimic the natural extracellular matrix. Herein we demonstrate a strategy for controlled, irreversible immobilization of a cell adhesion protein domain onto an otherwise bioinert substrate with well-defined protein orientation and density. Our approach uses a tri(ethylene glycol)-terminated self-assembled monolayer presenting a phosphonate ligand that is used to immobilize an engineered fusion protein. One component of the fusion protein, the 22 kDa serine esterase cutinase, reacts with the surface-bound ligand to form a site-specific covalent adduct, and the second component of the fusion protein is therefore immobilized on the surface. Here we use this approach to immobilize an engineered version of the 12 kDa 10th domain of fibronectin III (FnIII(10)Eng) to direct cell adhesion. Substrates presenting this protein mediated rapid attachment and spreading of Swiss 3T3 fibroblasts, while substrates presenting cutinase or the phosphonate ligand alone did not support cell attachment. In addition, we used Chinese hamster ovary cells engineered to express specific integrin receptors to show that FnIII(10)Eng interacts with multiple integrin cell surface receptors, including human alpha(V)- and alpha(5)-containing integrins. This general approach, in principle, can be used to immobilize any protein with an available gene sequence, providing an enabling technique for fundamental cell biology and tissue engineering.