The rotation of the benzoyl ring of methotrexate has been modeled in its complexes with dihydrofolate reductase (DHFR). The conjugate peak refinement method (Fischer, S.; Karplus, M. Chem. Phys. Lett. 1992, 194, 252) was used to generate conformational reaction paths and to locate transition states for the 180 degrees ring-flip process. The computed energy barriers for the 3’,5’-fluoro-substituted benzoyl ring of methotrexate (F(2)MTX) are 11.3 and 10.1 kcal/mol for the binary and ternary (with cofactor NADPH) complexes, respectively, which compare well with the experimental enthalpies of activation of 11.5 (binary) and 9.9 kcal/mol (ternary) from F-19-nmr spectroscopy (Clore, G. M.; Gronenborn, A. M.; Birdsall, B.; Feeney, J.; Roberts, G. C. K. Biochem. J. 1984, 217, 659). The pathways for the hydrogen-substituted isomer (H(2)MTX) are found to be similar, although the computed barrier heights are lower (6.5 and 5.0 kcal/mol, respectively). The process is characterized by an asynchronous transition of the two dihedral angles adjacent to the benzoyl ring and by a twin gating of the ring flip by four residues (Leu27, Phe30, Phe49, and Pro50), which form a "hydrophobic quadrant" around the ring. Perturbations of the protein up to 8 Angstrom from the active site (which expands by 1.6 Angstrom) make contributions to the energetics of the process. The local and global characteristics of the path and the effects of structural (crystallographic) solvent and the cofactor are discussed.