NAD(+)-dependent lactate dehydrogenase (LDH) is assembled onto a pyrroloquinoline quinone-NAD(+) monolayer. The redox active monolayer is assembled via covalent attachment of pyrroloquinoline quinone (PQQ) to a cystamine monolayer associated with a Au electrode, followed by covalent linkage of N-6-(2-aminoethyl)-NAD(+) to the monolayer. The surface coverage of PQQ and NAD(+) units is ca. 1.2 x 10(-10) moi cm(-2). The surface coverage of LDH bound to the redox active monolayer is ca. 3.5 x 10(-10) mol cm(-2). The assembled LDH monolayer is active in the bioelectrocatalytic oxidation of lactate. The bioelectrocatalyzed process involves the PQQ-mediated oxidation of the immobilized NADH in the presence of Ca2+ ions. The LDH associated with the PQQ-NAD(+) monolayer assembled on the electrode surface exhibits moderate stability, and the biocatalyst dissociates to the electrolyte solution. Dissociation of LDH is enhanced in the presence of solubilized NAD(+). Cross-linking of the monolayer-bound LDH with glutaric dialdehyde yields an integrated stable enzyme electrode for the bioelectrocatalyzed oxidation of lactate. The electrode acts as an amperometric biosensor for lactate. Affinity binding of NAD(+)-dependent alcohol dehydrogenase to the PQQ-NAD(+)-monolayer-modified Au electrode, followed by cross-linking of the enzyme, yields an enzyme electrode for the bioelectrochemical detection of ethanol.