Hydrophobins self assemble into amphipathic films at hydrophobic-hydrophilic interfaces. These proteins are involved in a broad range of processes in fungal development. We have studied the conformational changes that accompany the self-assembly of the hydrophobin SC3 with polarization-modulation IR reflection absorption spectroscopy, attenuated total reflection Fourier transform IR spectroscopy, and CD, and related them to changes in morphol. as obsd. by electron microscopy. Three states of SC3 have been spectroscopically identified previously as follows: the monomeric state, the a-helical state that is formed upon binding to a hydrophobic solid, and the b-sheet state, which is formed at the air-water interface. Here, we show that the formation of the b-sheet state of SC3 proceeds via two intermediates. The first intermediate has an IR spectrum indistinguishable from that of the a-helical state of SC3. The second intermediate is rich in b-sheet structure and has a featureless appearance under the electron microscope. The end state has the same secondary structure, but is characterized by the familiar 10-nm-wide rodlets. [on SciFinder (R)]