It has been suggested that the magnitudes of secondary kinetic isotope effects (2° KIEs) of enzyme-catalyzed reactions are an indicator of the extent of reaction-center rehybridization at the transition state. A 2° KIE value close to the corresponding secondary equilibrium isotope effects (2° EIE) is conventionally interpreted as indicating a late transition state that resembles the final product. The reliability of using this criterion to infer the structure of the transition state is examined by carrying out a theoretical investigation of the hybridization states of the hydride donor and acceptor in the Escherichia coli dihydrofolate reductase (ecDHFR)-catalyzed reaction for which a 2° KIE close to the 2° EIE was reported. Our results show that the donor carbon at the hydride transfer transition state resembles the reactant state more than the product state, whereas the acceptor carbon is more productlike, which is a symptom of transition state imbalance. The conclusion that the isotopically substituted carbon is reactant-like disagrees with the conclusion that would have been derived from the criterion of 2° KIEs and 2° EIEs, but the breakdown of the correlation with the equilibrium isotope effect can be explained by considering the effect of tunneling.