Silk fibroin fiber is a well-known textile, but is also used as biomaterial that is of interest for use in a variety of applications, usually in a hydrated state. Thus, the determination of the hydrated silk fibroin structure is important for understanding the function and in designing novel biomaterials. In this work, C-13 dipolar decoupling/magic angle spinning NMR was used to determine the local conformation of [3-C-13]Ala-, [3-C-13]Ser-, and [3-C-13]Tyr -Bombyx mori silk fibroin in a hydrated state. Ser residues are present predominantly in the crystalline domains, Tyr predominantly in the noncrystalline domains and Ala residues in both domains. The fraction of beta-sheet and two random coil distributions with fast and slow chain dynamics could be determined for all these residues by C-13 conformation-dependent chemical shift. The fraction of beta-sheet of these residues in the fiber and the crystalline fraction did not change significantly before and after hydration. The fraction of random coil conformations with fast motion in total random coil fraction of the hydrated fiber was 25%, 22%, and 11% for Ala, Ser, and Tyr residues, respectively. Thus, Tyr residues tend to hydrate relatively little among these residues. This information is the first detailed study of the effects of hydration on site-specific crystalline and noncrystalline domains of silk.