Isotopic labeling of Bombyx mori silk fibroin was achieved biosynthetically using two approaches. First, labeled fibroin was achieved by feeding silk worms [C-13(1)]Gly and [C-13(1)]Ala along with an artificial diet. Second, in vitro production of [N-15]Gly and [N-15]Ala labeled B. mori silk fibroin was accomplished by culturing the posterior silk glands isolated from five-day-old silkworm larvae in the fifth instar stage. Orientation-dependent N-15 and C-13 solid-state NMR spectra of these isotope-labeled silk fibroin fibers were observed when the fiber axis was arranged at various angles between 0 degrees and 90 degrees to the magnetic field direction. Such data from the silk II structure was simulated on the basis of the chemical shift anisotropy to determine the Euler angles that relate the principal axis system (PAS) to the fiber axis coordinate system. The dipolar modulated N-15 and C-13 chemical shift powder pattern spectra of C-13-N-15 double-labeled silk fibroin model peptides were observed and simulated to determine the Euler angles for transforming the PAS to molecular symmetry axis (MSA) system. The specific orientations of N-H, N-C-1, C-1=O, and C-1-N bonds for Ala and Gly residues in the oriented silk fibroin fiber were determined with a combination of these Euler angles. The conformational space for the Ala and Gly residues of the silk fibroin fibers was substantially reduced with these bond orientations and the known Calpha(i-1)-Calpha(i+1) vector orientation from fiber diffraction studies. The best fit torsion angles (phi,psi) within the reduced conformational space were determined as (-140 degrees, 142 degrees) and (-139 degrees, 135 degrees), respectively within experimental error (+/-5 degrees). The distance of the unit cell length determined here results in excellent agreement with the fiber diffraction data.