A transition structure for the hydride transfer step in dihydrofolate reductase has been characterized in hybrid quantum-mechanical/molecular-mechanical (QM/MM) calculations involving a fully flexible active-site region by means of the GRACE software. The results are compared with in vacuo calculations and the importance of including the environment effect is stressed. Thus, while a degeneracy endo and exo transition structure is obtained in the gas phase calculations, the protein environment selectively moulds the substrate in a conformation close to the exo transition structure. The enzyme compresses the substrate and the cofactor into a conformation close to the transition structure, thus facilitating the hydride transfer. The population analysis obtained by means of the in vacuo and the hybrid QM/MM methods renders similar net atomic charges for the donor and acceptor fragments, suggesting that a hydrogen more than a hydride ion is transferred. When che environment effect is included it allows the role of the Asp27 in stabilizing the cationic pteridine ring to be demonstrated, while the ordered structure of the X-ray crystallographic water molecules supports the hypothesis of an indirect transfer of a proton from Asp27 residue to the N5 atom of the substrate. A good agreement between experimental and QM/MM primary kinetic isotope effects is found. The comparison of the results obtained with the different methods and models shows that while some common features are observed, for reactions involving a large number of degrees of freedom it is not correct to refer to one unique transition structure. The transition state should be considered as an average of nearly degenerated transition structures.