From an analysis of NOEs observed in heteronuclear 3D NOE spectra of the [U-13C]ascomycin/FKBP complex, the conformation of ascomycin when bound to its putative target protein, FKBP, was determined. The conformation of ascomycin was found to be very different from the X-ray structure of FK-506 determined in the absence of FKBP, but more closely resembles that of uncomplexed rapamycin. In addition, from the observation of ascomycin/FKBP NOEs in the 3D spectra, those portions of ascomycin that are in close proximity to FKBP were identified, and they include parts of the piperidine, pyranose, and cyclohexyl rings. By using the structural information obtained from this study it may be possible to design ascomycin analogues that are structurally dissimilar from ascomycin but which nonetheless maintain the necessary functionality in the proper orientation for binding to FKBP. It is hoped that analogues could be designed with improved physicochemical properties and potentially lower toxicity, thus making them superior immunosuppressive agents. During the preparation of this manuscript, a report on the X-ray crystal structure of the FK-506/FKBP complex appeared. The conformation of ascomycin when bound to FKBP as determined by NMR is very similar to the conformation of FK-506 when bound to FKBP as determined by X-ray crystallography. In both structures of the bound ligands, the 9,10 amide bonds are trans and the relative orientation of the pyranose and cyclohexyl rings are the same. In the X-ray structure roughly 50% of the ligand surface is buried at the protein-ligand interface with the region around the allyl and cyclohexyl groups being exposed to solvent. We observed no NOEs from the ethyl group of ascomycin (analogous to the allyl group in FK-506) to the protein, which is consistent with this portion of the molecule being exposed to solvent. However, we do observe several NOEs from the cyclohexyl ring (Figure 5) to the protein, which clearly indicates that a portion of this ring is in close proximity to the protein in solution.