theta-Defensins are mammalian cyclic peptides that have antimicrobial activity and show potential as stable scaffolds for peptide-based drug design. The cyclic cystine ladder structural motif of ?-defensins has been characterized using NMR spectroscopy and is important for their structure and stability. However, the effect of the pronounced elongated topology of theta-defensins on their molecular motion is not yet understood. Studies of molecular motion by NMR relaxation measurements have been facilitated by the recent development of a semirecombinant method for producing cyclic peptides that allows for isotopic labeling. Here we have undertaken a multifield N-15 NMR relaxation analysis of the anti-HIV theta-defensin, HTD-2, and interpreted the experimental data using various models of overall and internal molecular motion. We found that it was necessary to apply a model that includes internal motion to account for the variations in the experimental T-1 and NOE data at different backbone amide sites in the peptide. Although an isotropic model with internal motion was the simplest model that provided a satisfactory fit with the experimental data, we cannot exclude the possibility that overall motion is anisotropic, especially considering the strikingly elongated topology of theta-defensins. The presence of flexible side chains, self-association, interactions with solvent, and internal motions are all potential contributors to the observed relaxation data. Internal motion consistent with the constraints imposed by the cyclic cystine ladder was observed in that the order parameters, S-2, show that residues in the turns are more flexible than those in the beta-sheet. This study provides insights into the dynamics of theta-defensins and information that might be useful in their application as scaffolds in drug design.