The high surface areas of nanostructured electrodes can provide for significantly enhanced surface loadings of electroactive materials. The fabrication and characterization of nanoporous gold (np-Au) substrates as electrodes for bioelectrochemical applications is described. Robust np-Au electrodes were prepared by sputtering a gold-silver alloy onto a glass support and subsequent dealloying of the silver component. Alloy layers were prepared with either a uniform or nonuniform distribution of silver and, post dealloying, showed clear differences in morphology on characterization with scanning electron microscopy. Redox reactions under kinetic control, in particular measurement of the charge required to strip a gold oxide layer, provided the most accurate measurements of the total electrochemically addressable electrode surface area, A(real). Values of A(real) up to 28 times that of the geometric electrode surface area, A(geo), were obtained. For diffusion-controlled reactions, overlapping diffusion zones between adjacent nanopores established limiting semi-infinite linear diffusion fields where the maximum current density was dependent on A(geo). The importance of measuring the surface area available for the immobilization was determined using the redox protein, cyt c. The area accessible to modification by a biological macromolecule, A(macro), such as cyt c was reduced by up to 40% compared to A(real), demonstrating that the confines of some nanopores were inaccessible to large macromolecules due to steric hindrances. Preliminary studies on the preparation of np-Au electrodes modified with osmium redox polymer hydrogels and Myrothecium verrucaria bilirubin oxidase (MvBOD) as a biocathode were performed; current densities of 500 mu A cm(-2) were obtained in unstirred solutions.