We have recorded (13) and N-15 high-resolution solid-state NMR spectra of Leu-enkephalin trihydrate (1, crystallized from aqueous solution), dihydrate (2, crystallized from aqueous methanol), -2H(2)O, 2DMF, X(unspecified solvent) (3, crystallized from aqueous N,N-dimethylformamide (DMF)), and Metenkephalin . 5.3H(2)O (4. crystallized from aqueous ethanol), at temperatures between 20 and -120 degrees C, to gain insight into the distinction of these crystalline polymorphs, backbone dynamics, and local conformational rearrangements induced by dehydration or freezing of motions of bound solvent molecules in the crystalline state. Four kinds of crystal forms revealed by X-ray diffraction studies were readily distinguished by C-13 NMR spectral patterns, and the number of nonequivalent chains in a unit cell was counted by either N-15 or C-13 split peaks. NR IR signals of two types of independent chains for 1 were resolved at 0 degrees C but were not distinguishable at ambient temperature owing to either two-sits exchange or phase transition. Up to four chains. however, were distinguished for 2-4 from the C-13 and (15) NMR splittings of the backbone C-alpha, C=O, N-H. COO-, and/or side-chain Tyr C-zeta signals. Noticeable spectral changes were induced at the C-13 NMR signals of the carboxyl group of the terminal Leu residue and Gly C-alpha by dehydration. It turned out that a distinct conformational change was accompanied with dehydration as manifested from the shortened interatomic distance between the C-13=O and N-15-H group. which is four bonds apart. In addition, pronounced spectral changes were noted when motions of bound solvent molecules in the crystals were frozen at lower temperatures. They were interpreted in terms of induced local conformational rearrangement, especially at the N- or C-terminus to which these solvent molecules were coordinated. We further examined the manner of the backbone and side-chain motions by means of the C-13 and proton spin-lattice relaxation times in the laboratory (T-1(C)) and rotating frames (T-1p(H)), respectively. In particular, the beta-bend structure of crystalline Leu-enkephalin trihydrate turned out to be very flexible in view of T-1(C) and T-1p(H) values as compared with the other crystalline forms taking the beta-sheet form. In contrast, there appears no such flexibility in Leu-enkephalin dihydrate despite a claimed similar beta-bend structure as determined by X-ray diffraction. It was also demonstrated that the presence or absence of side-chain motions was conveniently monitored by relative peak intensities of individual residues which were strongly influenced by the manner of local motions interfered with proton decoupling frequency. It was further shown that these molecular motions were also strongly affected by the bound solvent molecules.