Films made from polyheme cytochrome c(3) and the ionomer poly(ester sulfonic acid) AQ-29D or poly(styrenesulfonate) have been assembled by alternate layer-by-layer adsorption onto gold or silver electrodes. The build up of the multilayer structure has been monitored using quartz crystal microbalance and voltammetry techniques. AQ-29D polymer has been shown to form one stable monolayer film attached to metal electrodes. By using thus-modified-AQ-29D electrodes, successive layers of cytochrome c(3) and AQ-29D were deposited by alternate immersion in the respective solutions of protein and polymer. Alternate adsorption cycles have been repeated up to the construction of eight successive monolayers. Decreases in the coverage of redox active cytochrome on adsorption of the AQ-29D polymer layer on a sub-layer of cytochrome are observed. They can be explained on the basis of peel off of protein, or protein + AQ polymer, toward the solution. Several film construction strategies have been tested by varying the pretreatment of the metal electrode surface using positively or negatively charged species, or the nature of (monoheme or polyheme) cytochrome. AQ-29D films have been shown to exhibit the twofold role of polyanionomer and incorporating matrix, very promising for further development in biotechnologies. (C) 2003 Elsevier Science B.V. All rights reserved.