By using fully 15N- and 15N/13C-labeled Escherichia coli dihydrofolate reductase, the sequence-specific 1H and 15N NMR assignments were achieved for 95% of the backbone resonances and for 90% of the 13C alpha resonances in the binary folate complex. These assignments were made through a variety of three-dimensional proton-detected 15N and 13C experiments. A smaller but significant subset of side-chain 1H and 13C assignments were also determined. In this complex, only one 15N or 13C resonance was detected per 15N or 13C protein nucleus, which indicated a single conformation. Proton-detected 13C experiments were also performed with unlabeled DHFR, complexed with 13C-7/13C-9 folate to probe for multiple conformations of the substrate in its binary complex. As was found for the protein resonances, only a single bound resonance corresponding to a productive conformation could be detected for C-7. These results are consistent with an earlier report based on 1H NMR data [Falzone, C.J. et al. (1990) Biochemistry, 29, 9667-9677] and suggest that the E. coli enzyme is not involved in any catalytically unproductive binding modes in the binary complex. This feature of the E. coli enzyme seems to be unique among the bacterial forms of DHFR that have been studied to date.