Proton nuclear magnetic resonance spectroscopy has been used to detect two water molecules bound to residues in the active site of the Lactobacillus casei dihydrofolate reductase (DHFR). Their presence was detected by measuring nuclear Overhauser effects between NH protons in protein residues and protons in the individual bound water molecules in two-dimensional nuclear Overhauser effect spectroscopy (NOESY), in nuclear Overhauser effect spectroscopy in the rotating frame (ROESY) and three-dimensional 1H-15N ROESY-heteronuclear multiple quantum coherence spectra recorded on samples containing appropriately 15N-labelled DHFR. For the DHFR-methotrexate-NADPH complex, two bound molecules were found, one close to the Trp5 amide NH proton and the other near to the Trp21 indole HE1 proton: these correspond to two of the water molecules (Wat201 and Wat253) detected in the crystal structure studies described by Bolin and co-workers. However, the nuclear magnetic resonance experiments did not detect any of the other bound water molecules observed in the X-ray studies. The nuclear magnetic resonance results indicate that the two bound water molecules that were detected have lifetimes in the solution state that are longer than approximately two nanoseconds. This is of considerable interest, since one of these water molecules (Wat253) has been implicated as the likely proton donor in the catalytic reduction of dihydrofolate to tetrahydrofolate.