Gold nanoelectrode ensembles (NEEs) have been prepared by using an electroless plating method to deposit disk-shaped Au electrode elements (diameter = 38 nm) within the pores of a microporous polycarbonate template membrane. The electrochemistry of three electron-transfer mediators used for biosensors based on reductase enzymes - two phenothiazines (Azure A and B) and methylviologen - were investigated at these NEEs. As has been observed previously, detection limits obtained at the NEE are lower than corresponding detection limits for a Au disk electrode of conventional dimensions (diameter = 3.2 mm, called a macro electrode here). However, the enhancement in the detection limit at the NEE depends on the E-1/2 value of the mediator used. All three of these mediators have more negative E-1/2 values than the redox couples investigated previously at such NEEs. As such, their voltammetric waves are close to the negative limit for Au in the pH 7.4 buffer used as the electrolyte. The effects of background currents, associated with proton reduction, on the detection limits were investigated. At the macro electrode, the voltammograms for the phenothiazines are distorted by adsorption of the reduced forms to the electrode surface. This adsorption process is concentration dependent. The lower detection limits obtained at the NEE anew for the use of lower mediator concentrations, and this unwanted adsorption process can be eliminated at the NEE. Finally, we report here the first use of the NEEs for the determination of standard heterogeneous rates constants.