Gold surfaces with high biospecific affinity for pyridine-nucleotide-dependent dehydrogenases have been prepared on the basis of hydrophilic alkanethiol self-assembled monolayers bearing an artificial pyridine-nucleotide analogon as pendant ligand group. This site-specific bioaffinity binding provides a mild immobilization method to attach enzymes to metallic electrodes with a particular orientation which allows complexation of the ligand into the enzyme＇s NAD(+)-binding pocket. The investigation of protein adsorption events by surface plasmon resonance spectroscopy reveals that a satisfactorily high amount of protein (between 30 and 40%) of a densely packed protein layer can be tightly adsorbed on ligand-anchored alkanethiol self-assembled monolayers, whereas an appreciably lower amount adsorbs on their ligand-free analogous layers. The combination of surface plasmon resonance spectroscopy with electrochemistry ＇in situ＇ allows us to monitor simultaneously the amount and electrocatalytic activity of the immobilized biological material. The ratio (K) between the protein＇s electroenzymatic response and the average thickness of the immobilized protein layer can be taken as an indication of the state of the protein on the electrode surface. It was found from these measurements that electrodes modified with a monolayer of lactate dehydrogenase lose electroenzymatic activity upon successive shots of lactate due to progressive detachment of the protein from the ligand-anchored monolayer. The catalytic activity of the enzyme, however, remains unaltered during the electrochemical runs, as shown by the constant calculated K values (K approximate to 1.0 mu A cm(-2) nm). The ratio obtained between K values for enzymatic electrodes prepared from ligand-anchored and ligand-free alkanethiol monolayers suggests that lactate dehydrogenase is anchored to the ligand monolayer through two NAD(+)-binding pockets which are not involved in the electroenzymatic reaction.