The hydrophobin Sc3 from the fungus Schizophyllum commune assembles from the aqueous phase into ordered structures with substantially different characteristics depending upon experimental conditions. Under the first condition, a vortexing procedure widely reported in the literature, interfacial assembly yields highly ordered, stacked beta-sheets. We have also observed a previously unreported assembly of Sc3 under a second condition, which occurs in a time-dependent manner from quiescent solution. The resulting types of assembled states have been compared utilizing fluorescence techniques, sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, density gradient centrifugation, and phase contrast and atomic force microscopy. A model based on this study and previous literature is proposed that suggests three distinct states of Sc3: (1) soluble Sc3 consisting of unimers or multimers. in micelle-like association, (2) interfacially assembled I-Sc3 with highly ordered, stacked beta-sheets, presumably formed in a templated manner at the air/water interface of microscopic bubbles generated by vortexing, and (3) solution-assembled S-Sc3, a less-ordered structure formed in a time-dependent manner in the absence of an interface.