The X-ray crystal structure of 28-O-methylrapamycin 2 bound to FKBP12 is described. This rapamycin analogue binds to FKBP12 with an affinity comparable to that of rapamycin, but its immunosupressive activity is reduced by a factor of 1000. The atomic structure of the complex formed by FKBP12 and 28-O-methylrapamycin is compared with those of the FKBP12-rapamycin and FKBP12-FK506 complexes. The steric 28-O-methyl group induces a dramatic shift in the orientation of the cyclohexyl moiety, which is now in a position similar to the one observed for the cyclohexyl subunit in the FKBP12-FK506 complex. The conformation of the macrocyclic part of the molecule remains unchanged. As a consequence of 28-O-methylation and the resulting modified orientation taken by the cyclohexyl subunit, two intermolecular hydrogen bonds between the ligand and the binding protein are lost in comparison to the FKBP12-rapamycin complex. The affinity for FKBP12 is not significantly affected by these structural changes, but the immunosuppressive activity is greatly reduced, suggesting a critical role for the cyclohexyl ring in the interaction of the FKBP12-rapamycin complex with its molecular target.