The effects of coenzyme binding on the seven histidine C2 proton resonances of Lactobacillus casei dihydrofolate reductase have been determined. Binary complexes containing NADP+, NADPH, and their hypoxanthine, thionicotinamide, and acetylpyridine analogues, together with ternary complexes containing the inhibitors trimethoprim or methotrexate, have been examined. Four of the histidine residues are affected by coenzyme binding. The largest effect-a marked upfield shift (0.85 ppm) of the C2 proton resonance-is seen for His-64. The hypoxanthine analogue of the coenzyme was found to produce a smaller upfield shift and, in addition, a decrease in the pK of His-64. The effects on this reductase are discussed in the light of the crystal structure [Matthews, D. A., Alden, R. A., Bolin, J. T., Filman, D. J., Freer, S. T., Hamlin, R., Hol, W. G. J., Kisliuk, R. L., Pastore, E. J., Plante, L. T., Xuong, N., & Kraut, J. (1978) J. Biol. Chem. 253, 6946], and it is concluded that His-64 is close to a carboxyl group in the free enzyme and that the hypoxanthine ring binds in a somewhat different orientation to the adenine ring. The effects on histidine resonances A, E, and G are significantly different for oxidized and reduced coenzymes. The changes in pK of the histidines giving rise to resonances A and E (probably His-22 and His-18) are discussed in terms of ligand-induced conformational changes, which differ for NADP+ and NADPH.