A cDNA encoding dihydropyrimidinase has been isolated from a rat cDNA library. The N-terminal and an internal amino acid sequences were determined, and PCR primers were designed based on these sequences. Using a cDNA fragment amplified by RT-PCR with these primers, three cDNA clones were isolated from a rat liver library. The clone with the longest insert of 2129 bp contained a 1557 bp open reading frame encoding a polypeptide of 519 residues with a molecular mass of 56 832 Da.