Surface-modified gold electrodes with biospecific affinity for NAD(H)-dependent lactate dehydrogenase (LDH) have been prepared from mixed self-assembled monolayers bearing the coenzyme-analogous, Cibacron Blue 3FG-A, as anchored pendant group. Enzymatic monolayers are formed by formation of a complex upon the site-specific binding of Cibacron Blue to the enzyme through its NAD(+)-binding pocket. In such monolayers the enzyme may be expected to lie oriented on the electrode surface with its NAD(+)-binding pecker (and therefore its active site) connected directly to the metal through a spacer. However, since Cibacron Blue is not electroactive within the range of potentials used to electro-oxidize lactate, the ligand monolayer was found not to be involved in the electroenzymatic reaction, but worked only as an anchoring system for the enzyme. LDH-modified electrodes were able to electro-oxidize lactate at small overpotentials only in the presence of soluble coenzyme, NAD(+). The electroenzymatic activity of LDH-modified electrodes depended on the amount of enzyme immobilized and on the concentration of lactate in the reaction mixture, with a Michaelis constant K-M = 10mM for lactate. The overall electroenzymatic activity of LDH-modified electrodes was found to arise from both, biospecifically and unspecifically, adsorbed enzyme. Whereas the amperometric response due to the biospecifically adsorbed species dropped off following the first shot of lactate, that due to unspecifically adsorbed enzyme remained stable after several electro-oxidation runs and only disappeared after inactivation of the enzyme.