Dynamic mechanical analysis (DMA) was applied to investigate the correlation between dynamic mechanical behaviors of regenerated silk fibroin (RSF) and its structural transition which was characterized by near-infrared (NIR) spectroscopy. The tan delta peak split of DMA demonstrated that the apparent glass transition of amorphous RSF film (at around 177 degrees C) was the contribution of both uncrystallizable and crystallizable segments through homogeneous amide-amide hydrogen bonds, which were gradually separated from each other and produced disordered and beta-sheet domains during ethanol treatment. Furthermore. DMA was also applied as "thermal fingerprint" to investigate water effect on the disordered domains of silk fibroin. The results showed that glass transition temperature of permanently disordered domains in crystallized RSF films was increased from 155 degrees C to 190 degrees C after thermal dehydration, and even approached 205 degrees C for stretched RSF films. (C) 2010 Elsevier Ltd. All rights reserved.