Overlapping cDNAs encoding dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme of pyrimidine degradation, were isolated from a rat liver cDNA library, and a 4,353 bp cDNA sequence with a 3,075 bp open reading frame encoding a polypeptide of 1,025 residues with a molecular mass of 111,458 Da was determined. Western blot analysis of COS-7 cells that were transfected with an RT-PCR-derived DPD cDNA showed that the product from the cDNA binds to an anti-serum for DPD. Northern blot analysis was performed by using a partial fragment of the cloned cDNA as a probe. The mRNA level, the protein level, and the activity of DPD in the rat liver increased to the adult level by 3 mo of age. The levels of DPD in the liver of adrenalectomized rats increased 48 h after glucocorticoid administration. The increase of DPD after birth may be caused by the increase in glucocorticoid level.