Interfacial investigations of a protein monolayer electrochemical system, equine cytochrome c (cyt C) adsorbed to carboxylic acid terminated self-assembled monolayer (COOH SAM) modified gold electrodes, were performed. Electrochemical, spectroscopic, and scanning probe microscopy techniques were utilized to explore the influence of gold topography in the cyt c/COOHSAM/gold system. COOHSAMs were prepared from 14-mereaptotetradecanoic acid and 11-mereaptoundecanoic acid on a variety of substrates including evaporated, bulk, single crystal, and epitaxially grown gold on mica. These substrates encompassed a wide range of surface roughness. As the topography of the gold became smoother, SAMs exhibited an increased ability to block a solution probe molecule, indicative of a lower level of defectiveness. At the same time, after exposure to equine cyt c deposition solutions, the extent of adsorption and the magnitude of the electrochemical response of the adsorbed cyt c decreased significantly with increasingly smooth substrates. The results show cyt c adsorption and electrochemistry to be intimately related to the density of defects in the SAM, which in turn are largely dictated by the topography of the gold substrate. This hypothesis is supported by experiments in which the density of defects in the SAMs was controlled on each type of gold substrate using intentional roughening/smoothing procedures as well as through the use of mixed SAMs. Results are interpreted in terms of the topographically dependent acid/base properties of the COOH SAMs, which can limit the electrostatically driven adsorption of cyt c and the effectiveness of the protein's electronic coupling at the differently textured SAM surfaces.