All the aromatic proton resonances in the 500-MHz NMR spectra of Lactobacillus casei dihydrofolate reductase have been assigned for several of its complexes with inhibitors. For the complexes with methotrexate and trimethoprim this was achieved by using a combination of NMR techniques in conjunction with a selectively deuterated protein designed to simplify the spectra such that nuclear Overhauser effect (NOE) connections could be detected with greater ease and certainty. By correlating these NOE data with crystal structure data on related complexes it was possible to assign all the aromatic resonances and to extend these assignments to spectra of other complexes of dihydrofolate reductase. The conformation-dependent chemical shifts observed for many of the resonances could be explained qualitatively, but not quantitatively, in terms of ring-current shifts. The discrepancies between calculated ring-current shifts and the observed conformation-dependent shifts could not in general be accounted for satisfactorily in terms of carbonyl-group anisotropic shielding contributions calculated using presently available models. In the case of the H delta 1, delta 2 protons of Phe30 some of the discrepancy probably results from a difference in the conformation of the Phe ring between the solution and crystal states.