Dihydrofolate reductase (DHFR) is a household enzyme that is responsible for the NADPH-dependent conversion of dihydrofolate (DHF) to tetrahydrofolate (THF).The mechanism involves proton donation at N5 and hydride transfer at C6 on the substrate.Although the X-ray structure of the pseudo-Michaelis ternary complex DHFR-folate-NADP+ is available, the inability of X-rays to determine the protonation states of the active site residues and of substrate has led to the lack of consensus on a catalytic mechanism.To resolve this ambiguity, we conducted neutron and ultrahigh resolution X-ray crystallographic studies of the E.coli DHFR-folate-NADP+ complex.