We have studied the hydride transfer reaction catalyzed by the enzyme dihydrofolate reductase (DHFR) and the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH); the substrate is 5-protonated 7,8-dihydrofolate, and the product is tetrahydrofolate. The potential energy surface is modeled by a combined quantum mechanical-molecular mechanical (QM/MM) method employing Austin model 1 (AM1) and a simple valence bond potential for 69 QM atoms and employing the CHARMM22 and TIP3P molecular mechanics force fields for the other 21 399 atoms; the QM and MM regions are joined by two boundary atoms treated by the generalized hybrid orbital (GHO) method. All simulations are carried out using periodic boundary conditions at neutral pH and 298 K. In stage 1, a reaction coordinate is defined as the difference between the breaking and forming bond distances to the hydride ion, and a quasithermodynamic free energy profile is calculated along this reaction coordinate. This calculation includes quantization effects on bound vibrations but not on the reaction coordinate, and it is used to locate the variational transition state that defines a transition state ensemble. Then, the key interactions at the reactant, variational transition state, and product are analyzed in terms of both bond distances and electrostatic energies. The results of both analyses support the conclusion derived from previous mutational studies that the M20 loop of DHFR makes an important contribution to the electrostatic stabilization of the hydride transfer transition state. Third, transmission coefficients (including recrossing factors and multidimensional tunneling) are calculated and averaged over the transition state ensemble. These averaged transmission coefficients, combined with the quasithermodynamic free energy profile determined in stage 1, allow us to calculate rate constants, phenomenological free energies of activation, and primary and secondary kinetic isotope effects. A primary kinetic isotope effect (KIE) of 2.8 has been obtained, in good agreement with the experimentally determined value of 3.0 and with the value 3.2 calculated previously. The primary KIE is mainly a consequence of the quantization of bound vibrations. In contrast, the secondary KIE, with a value of 1.13, is almost entirely due to dynamical effects on the reaction coordinate, especially tunneling.