Dihydrofolate reductase (DHFR) is a small, monomeric enzyme that catalyzes the conversion of dihydrofolate to tetrahydrofolate, a step crucial to the normal biosynthesis of purines, pyrimidines and amino acids. Its small size and well-established mechanism make it an ideal subject for investigations into the impact of enzyme motion on catalysis. Due to the kinetic complexity of this catalytic pathway, intrinsic kinetic isotope effects (KIEs) have proven to be highly sensitive probes of the hydride transfer step (the chemical step). Primary (1°) and secondary (2°) KIEs for the wild-type (WT) enzyme have previously been studied using mixed-labeling experiments, and the same method has been used to determine intrinsic 1° KIEs for a series of mutants: G121V, M42W, and the double mutant, G121V-M42W. Intrinsic 2° KIEs for the G121V mutant were also determined. The temperature dependence data for the intrinsic 1° KIEs support a theoretical model involving environmentally coupled quantum tunneling at the hydride transfer step. The 2° H/T and D/T KIE's indicated that there is no coupled motion between the 1° hydrogen (that being transferred) and the 2° (geminal hydrogen). In the current study, 2° H/T KIEs were measured for the M42W and the double mutant. The intrinsic 2° KIEs were compared to those values previously obtained for the WT and the G121V mutant. The collected 2° KIE data can now be used to draw conclusions about coupled motion in DHFR, and to parameterize the DHFR reaction potential surface in computational studies.