The binding of the FK506/FKBP-12 complex to calcineurin (CN), its putative target for immunosuppression, involves recognition of solvent-exposed regions of the ligand as well as FKBP-12 residues near the active site. The R42K, H87V double mutation of FKBP-12 decreases the CN affinity of the complex by 550-fold [Aldape, R. A., Futer, O., DeCenzo, M. T., Jarrett, B. P., Murcko, M. A., & Livingston, D. J. (1992) J. Biol. Chem. 267, 16029-16032]. This work reports the solution structure of 13C-labeled FK506 bound to R42K, H87V FKBP-12. Assignments and NOE measurements at three mixing times were made from inverse-detected 1H-13C NMR experiments. Structures were calculated by several different methods, including distance geometry, restrained molecular dynamics, and molecular dynamics with time-averaged restraints. The NMR structures of the ligand are very well defined by the NOE restraints and differ slightly from the X-ray structure in regions that are involved in crystal packing. Comparison with the NMR structure of FK506 bound to wild-type FKBP-12 reveals that the R42K, H87V mutation causes the ligand backbone near C16 to move by 2.5 to 4.5 A, reorients 15-MeO by 90 degrees, and shifts 13-MeO by approximately 1.5 A. FK506 appears to undergo a concerted, mutationally induced shift in the binding pocket, with the greatest changes occurring in the effector region of the drug. The altered effector conformation of mutant-bound FK506 may perturb interactions between the drug and CN, thus accounting for the effect of the double mutation upon the CN inhibitory activity of the complex.