Optimal conditions for the assay of dihydropteridine reductase in crude tissue extracts have been developed. Among adult rat tissues, liver and kidney had the highest activity, followed by thymus, lung, cerebellum and cerebrum. Fetal tissues and neoplasms exhibit lower activities than the cognate adult tissues. In both liver and kidney the developmental upsurge of dihydropteridine reductase, which produces the cofactor necessary for the hydroxylation of aromatic amino acids, begins earlier than does that of phenylalanine hydroxylase. In human liver and lung also, the dihydropteridine reductase activity doubles between midgestation and adult life. The undifferentiated enzymic composition of neoplasms is reflected by the lower dihydropteridine reductase content of pulmonary tumors than of nonneoplastic human lung.