The overall objective of the study was to understand the interplay between protein structure and function for biologically important macromolecules using the enzyme dihydropteridine reductase (DHPR) as a model system. The enzyme DHPR is found in the brain where it participates in the reproduction of tetrahydrobiopterin from the NADH-mediated reduction of quinonoid dihydrobiopterin, an essential cofactor in the biosynthetic reactions of phenylalanine, tyrosine, and tryptophan. In recent researches, the x-ray structure of the apoenzyme and the cofactor bound enzyme was determined. However, the crystal structure of the enzyme together with its substrate and cofactor are yet to be solved. We compared the fluorescence characteristics of the cofactor and/or inhibitor when complexed with DHPR. Data revealed that a pronounce change in fluorescence intensity and fluorescence peaks results when the small molecules studied were complexed with DHPR. The presence of DHPR quenches the fluorescence of the cofactor, NADH, and the inhibitor trimethoprim. For the TMP complexes, measurements at different temperatures revealed that the rate of temperature dependent fluorescence quenching were greater in the ternary complex than in the unbound TMP. One intriguing result was that for one inhibitor, 6,7-dimethyl 5,6,7,8 tetrahydrobiopterin, the presence DHPR results in an enhancement of fluorescence. It is clear from our results that small molecules interact with DHPR and it appears that the inhibitors interact with DHPR even in the absence of the cofactor, NADH.