The ethanol-induced conformation transition of regenerated Bombyx mori silk fibroin membrane from a poorly defined to the well ordered state was monitored by time-resolved Fourier transform infrared spectroscopy (FTIR) for the first time. From the analysis of FTIR difference spectra, taken on time scales as short as 6 s and up to 1 h after addition of ethanol, intensity vs. time plots of an increasing band at 1618 cm(-1) were observed indicating formation of a beta-sheet coincident with the loss of intensity of a band at 1668 cm(-1) indicating decreases of random coil and/or silk I structure. Both infrared markers were fitted with identical biphasic exponential decay functions, however, there was a clear burst phase occurring prior to the onset of the observed transitions. The conformation transition process is indicated to either proceed sequentially through (at least) two intermediate states that contain different levels of beta-sheet structure or to have parallel pathways of initial beta-sheet formation followed by a slower 'perfection' phase. The first observed process forms in a burst phase a few seconds after mixing (or even faster), prior to the collection of the first spectrum at 6 s. The second observed process occurs with a time constant of approximately 0.5 min, the intermediate present at this stage then continues with a time constant of 5.5 min completing the observed formation of the beta-sheet. The conformation transition of this slower intermediate is not only indicated by an analysis of the kinetics of the random coil and beta-sheet-specific bands discussed above, it roughly coincides with the appearance of an additional infrared marker at 1695 cm(-1), which may be a marker for beta-sheet structure specific to the formation of the perfected structure. The conformation transition of this protein analyzed by infrared spectroscopy provides insight into a part of the fascinating process of cocoon formation in B. mori.