The conformation and dynamics of a uniformly C-13-labeled glycolipid, digalactosyldiacylglycerol (DGDG), is studied in a membrane environment using NMR spectroscopy. Dipolar couplings and C-13 chemical shift anisotropy offsets which appear in magnetically oriented phospholipid-based membrane fragments are used to derive a detailed description of the conformation and amplitudes of motion. C-13 relaxation rates (T-1, T-2, and NOE) were then measured for C-13 sites in DGDG anchored to isotropically tumbling phospholipid bilayers. Using a Lipari and Szabo type approach, a nonlinear least squares fit of relaxation rates to motional parameters yielded rates and amplitudes of internal motions for the first and terminal sugar of the,glycolipid head group. Generalized order parameters extracted from relaxation data show good agreement with order parameters used to fit dipolar couplings. The combined model is analyzed in terms of potential energy maps generated using a version of AMBER modified to include a membrane interaction energy term. The experimental conformational preferences of the terminal alpha(1-6) linkage in DGDG agree quite well with predictions based on these calculations.