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We present results concerning the formation of Langmuir−Blodgett (LB) films of a class I hydrophobin from Pleurotus ostreatusat the air−water interface, and their structure as Langmuir−Blodgett (LB) films when deposited on silicon substrates. LB films of the hydrophobin were investigated by atomic force microscopy (AFM). We observed that the compressed film at the air−water interface exhibits a molecular depletion even at low surface pressure. In order to estimate the surface molecular concentration, we fit the experimental isotherm with Volmerʼs equation describing the equation of state for molecular monolayers. We found that about 1/ 10of the molecules contribute to the surface film formation. When transferred on silicon substrates, compact and uniform monomolecular layers about 2.5 nm thick, comparable to a typical molecular size, were observed. The monolayers coexist with protein aggregates, under the typical rodlet form with a uniform thickness of about 5.0 nm. The observed rodlets appear to be a hydrophilic bilayer and can then be responsible for the surface molecular depletion. [ABSTRACT FROM AUTHOR]