The use of an alkanethiol-based self-assembled mixed monolayer as an electronic relay system effecting mediated electron transfer between immobilized glucose oxidase (GOx) and a gold electrode is reported. We comp are the behavior of mixed monolayers of various compositions of 16-ferrocenylhexadecanethiol (16FAT) and aminoethanethiol, to which GOx is attached, as biosensors for glucose. The amperometric response of such electrodes in the presence of glucose in solution depends on the mole ratio between the 16FAT molecules and the attached protein molecules. The most sensitive system is a mixed monolayer that contains 7% 16FAT. For higher 16FAT concentrations, both a catalytic response and a wave corresponding to reversible 16FAT voltammetry are observed in the presence of glucose. This suggests that there are separate domains of 16FAT and of aminoethanethiol in such a mixed monolayer. When the mixed monolayer contains more than 7% 16FAT, a portion of the 16FAT molecules cannot "feel" the GOx and does not function as relays. The existence of these domains was also characterized by studying the solution voltammetry of Ru(NH3)(6)(3+) at electrodes with various proportions of 16FAT and amino-ethanethiol.