Controlling the molecular orientation of protein molecules immobilized on insoluble substrates is now recognized as an important issue in numerous fields such as biosensing. In this study, we have investigated adsorption of horse heart cytochrome c (hcyt c) to a silane-based, self-assembled monolayer (SAM) as a strategy for preparing an oriented protein film. Previous attempts in our laboratory to create oriented protein films using this strategy were unsuccessful because multiple types of adsorptive interactions between the protein and the SAM produced a broad range of adsorbed orientations. In contrast, here we show that when a single type of site-directed interaction between hcyt c molecules and the SAM predominates, an oriented protein film results. Sulfonate-terminated SAMs were prepared by in situ oxidation of thioacetate SAMs. Electrostatically mediated adsorption of positively charged hcyt c produced a film with a porphyrin tilt angle distribution of 41degrees +/- 13degrees, which is significantly narrower than distributions measured previously for cytochrome c films formed on other types of silane-based SAMs.