We have scaled down electrochemical assays of redox-active enzymes enabling us to study small numbers of molecules. Our approach is based on lithographically fabricated Au nanoelectrodes with dimensions down to ca. 70 x 70 nm(2). We first present a detailed characterization of the electrodes using a combination of scanning electron microscopy, cyclic voltammetry, and finite-element modeling. We then demonstrate the viability of the approach by focusing on the highly active [NiFe]-hydrogenase from Allochromatium vinosum immobilized on polymyxin-pretreated Au. Using this system, we successfully demonstrate a distinct catalytic response from less than 50 enzyme molecules. These results strongly suggest the feasibility of using bioelectrochemistry as a new tool for studying redox enzymes at the single-molecule level.