The buildup of layer-by-layer (LBL) assemblies of a series of acid proteins and a positively charged polyelectrolyte, Poly (L-lysine) (PLL), has been carried out onto gold surfaces modified through self-assembled alkanethiol. The stability of the assemblies was examined using quartz crystal microgravimetry (QCM) and electrochemical techniques. LBL assemblies have also been reported in the case of alternate adsorption of acid proteins (such as ferredoxin) and basic protein (such as cytochrome c(3) from Desulfovibrio vulgaris Hildenborough). The stability of the deposited layers has been shown to be improved in the presence of salt (NaCl). LBL film construction strategy has been used for the construction of alternate layers of hydrogenase, a slightly acid protein, and of its physiological partner, cytochrome c(3). The catalytic efficiency of immobilized hydrogenase has been evaluating by measuring the catalytic current observed in the presence of an artificial shuttle, methylviologen. Despite release phenomena, it has been established that both redox physiological partners can be co-immobilized in piled up layers. (C) 2004 Elsevier B.V. All rights reserved.