Activity differences of the two amide protons of biotin were revisited via perspective from proton transfer in the gas phase, employing the B3LYP/6-31G* methods. Our results reveal that for different conformation of biotin, the activity of the two amide is different: the 3NH proton was more active than that of 1-NH for extended biotin, while less active for folded conformation, which indicated that the 1-NH proton of biotin is not always more active than the 3-NH proton. The results here may be helpful for realizing the mechanisms of activity differences of the two NH protons in solution. (c) 2006 Elsevier B.V. All rights reserved.