In contrast to previous reports, fluorescence and ultraviolet difference spectroscopy are shown to reflect the interaction of the inhibitor, 5-fluoro-2'-deoxyuridylate, with thymidylate synthetase isolated from amethopterin-resistant Lactobacillus casei. Analysis of the quenching of protein fluorescence upon nucleotide binding yields a value of 2.1 X 10(5) M-1 for the association constant for binary complex formation. The ultraviolet difference spectrum for the nucleotide-enzyme complex exhibited a broad trough with the greatest loss of absorbance centered at 275 nm. Results of previous circular dichroic studies of nucleotide-enzyme binary complexes showed apparently parallel changes in ellipticity in the 267-269-nm and 290-nm regions, which were interpreted to reflect coordinated alteration in nucleotide and enzyme structure, respectively. When examined by difference circular dichroic spectroscopy, the nucleotide-enzyme interaction is accompanied by a substantial loss in ellipticity from 250 to 300 nm which is greatest at 280 nm. We interpret the results obtained from the three light spectroscopic techniques as indications of subtle alterations in the environments of certain tyrosine and tryptophan residues in the enzyme which are caused by the association of 5-fluoro-2'-deoxyuridylate with the enzyme.