The heterogeneous electron-transfer properties of flavin adenine dinucleotide (FAD) were studied at self-assembled monolayer (SAM)-modified gold electrodes and compared to ferrocene-modified SAMs as a reference system. A modified form of FAD, N-6-(2-aminoethyl)-FAD, was covalently immobilized onto a mixed self-assembled monolayer (SAMs) formed by two series of alkanethiols (HS-(CH2)(n)-COOH with a diluent HS-(CH2)(n)-CH3 where n = 5, 7, 10, or 15 and HS-(CH2)(n)-COOH with a diluent HS-(CH2)(n-)CH2OH where n = 5, 7, 10, or 11). The electron-transfer (ET) rates followed an exponential decay with the increasing thickness of the SAM (k(et) = A exp(-betan); n = number of bonds) with an attenuation factor (beta) of 1.0 per bond for FAD with both diluents. Despite the similarity in beta the apparent rate constant for electron transfer was 2 orders of magnitude greater with the alcohol-terminated diluent relative to the methyl-terminated SAM. In comparison the ferrocenyl SAMs also had beta values of 1.0-1.2 per bond, depending on the composition of the diluent layer, with the apparent rate constant for electron transfer being significantly faster with the alcohol-terminated diluent layer. The reconstitution of apo-glucose oxidase (apo-GOx) onto a FAD-modified gold electrode was also carried out; however, no biocatalytic activity was observed. Based on the beta value for the FAD-modified electrodes and the magnitude of the apparent rate of electron transfer, rate constants for direct electron transfer to glucose oxidase at carbon nanotube-modified electrodes and graphite electrodes suggest the enzyme has been partially denatured from its native configuration, thus bringing the redox-active center of the enzyme closer to the electrode and allowing appreciable ET to be observed.