The adsorption behaviour of five different globular proteins on pure silicon substrates and on preadsorbed polyampholytic monolayers has been investigated as a function of protein concentration. The prelayers were prepared by adsorption of the ampholytic diblock copolymer poly(methacrylic acid)-block-poly((dimethylamino)ethyl methacrylate) (PMAA-b-PDMAEMA). This polyampholyte adsorbs in densely packed micelles directly from aqueous solution. Ellipsometry was used to determine the amount of adsorbed polyampholyte and protein. While ATR-IR spectroscopy gives information about the adsorption and desorption behaviour of the preadsorbed polyampholytic layer, the lateral structures of the dried films were investigated by scanning force microscopy (SFM). The amount of protein adsorbed was found to be strongly influenced by the preadsorbed polyampholyte compared to the adsorption on the pure silicon substrates. No displacement of the polyampholyte by the proteins was detected. In most cases the protein adsorption was reduced by the preadsorbed polyampholytic layer. The observed trends are explained by the change in electrostatic and hydrophilic characteristics of the substrates. Furthermore, the entropy of adsorption has to be taken into account.