Atomic force microscopy (AFM) has been used to study the fungal hydrophobin protein HYPA produced by the mushroom Agaricus bisporus. The protein was adsorbed from dilute solution onto highly oriented pyrolytic graphite (HOPG), a hydrophobic and atomically flat substrate, and the resulting self-assembled layers were imaged under n-butanol. The nature of the self-assembled layer was found to be influenced by the protein concentration in the solution. At relatively high protein concentrations (20 mu g mL(-1)) the monolayer formed contained randomly oriented protein molecules. However, at lower protein concentrations (2 mu g mL(-1)) a highly ordered monolayer was formed with a higher level of surface coverage. The protein molecules appear to assemble end-to-end to form short rods with an average length of 80 nm, The thickness of the ordered monolayer is uniform and around 3.6 nm, Finally, the effect of washing the adsorbed protein layers with solutions of sodium dodecyl sulphate, leading to partial removal of the HYPA monolayer and bilayer formation, and of washing with trifluoroacetic acid which strongly disrupts the adsorbed monolayer, were also examined.