Nine single genetic mutants of rat dihydropteridine reductase (EC 126.96.36.199), D37I, W86I, Y146F, Y146H, K150Q, K150I, K150M, N186A, and A133S and one double mutant, Y146F/K150Q, have been engineered, overexpressed in Escherichia coli and their proteins purified. Of these, five, W86I, Y146F, Y146H, Y146F/K150Q, and A133S, have been crystallized and structurally characterized. Kinetic constants for each of the mutant enzyme forms, except N186A, which was too unstable to isolate in a homogeneous form, have been derived and in the five instances where structures are available the altered activities have been interpreted by correlation with these structures. It is readily apparent that specific interactions of the apoenzyme with the cofactor, NADH, are vital to the integrity of the total protein tertiary structure and that the generation of the active site requires bound cofactor in addition to a suitably placed W86. Thus when the three major centers for hydrogen bonding to the cofactor are mutated, i.e. 37, 150, and 186, an unstable partially active enzyme is formed. It is also apparent that tyrosine 146 is vital to the activity of the enzyme, as the Y146F mutant is almost inactive having only 1.1% of wild-type activity. However, when an additional mutation, K150Q, is made, the rearrangement of water molecules in the vicinity of Lys150 is accompanied by the recovery of 50% of the wild-type activity. It is suggested that the involvement of a water molecule compensates for the loss of the tyrosyl hydroxyl group. The difference between tyrosine and histidine groups at 146 is seen in the comparably unfavorable geometry of hydrogen bonds exhibited by the latter to the substrate, reducing the activity to 15% of the wild type. The mutant A133S shows little alteration in activity; however, its hydroxyl substituent contributes to the active site by providing a possible additional proton sink. This is of little value to dihydropteridine reductase but may be significant in the sequentially analogous short chain dehydrogenases/reductases, where a serine is the amino acid of choice for this position.