Sc3p is the major hydrophobin produced and excreted by the wood-rotting fungus Schizophyllum commune when grown as a monokaryon in culture. In addition to Sc3p, several lower molecular weight proteins of unknown function and a high molecular weight polysaccharide, schizophyllan, are present in this fungal culture medium. Previous studies in our laboratory indicated that schizophyllan and hydrophobin, as produced in the culture supernatant, work in concert to ultimately produce stable films on hydrophilic substrates. We report a new procedure for isolation of hydrophobin that does not require treatment with strong organic acids. This procedure allows a systematic evaluation of the importance of the individual hydrophobin and schizophyllan components in solution stabilization and film formation from a conformational state more representative of that found during fungal production. Experimental results indicate that schizophyllan stabilizes small hydrophobin multimers in aqueous solution. In addition, schizophyllan is necessary for subsequent hydrophobin assembly into films on hydrophilic surfaces. By contrast, hydrophobin alone is sufficient for thin film assembly onto a hydrophobic surface; however, hydrophobin aggregation at high protein concentration in the absence of schizophyllan may limit the efficiency of film formation. A model has been proposed in which the polysaccharide acts as a hydrophilic stabilizer of hydrophobin in solution preventing self-aggregation. The schizophyllan-stabilized complex may also allow conformational mobility for lateral reorganization during deposition and film coalescence.