A cDNA encoding β-ureidopropionase (BUP) Was isolated from a human liver cDNA library, expressed in E. coil, and purified from the culture extract. The 2, 006 by cDNA contained a 1, 152 by open reading frame encoding a protein of 384 amino acids with a mo-lecular weight of 43, 165 Da. The subunit molecular weight of the enzyme expressed was about 43, 000 Da. The enzyme was inhibited by 1 mM propionate, but not by 10 mite β-ala-nine. Chemical analysis of the purified human BUP showed 0.54 zinc atoms per subunit, and the sequence of BUP cDNA contained one putative zinc-binding site motif. The purified enzyme had a pI of 565, and exhibited positive cooperativity with N-carbamoyl-β-alanine as the substrate with a Hill coefficient 2.0. These properties of human BUP, except the inhi-bition by β-alanine, were similar to the rat liver purified enzyme. β-Alanine inhibits rats BUP activity. The complex regulatory function and the negative cooperative mechanism of BUP by β-alanine have been observed in rats. This kind of mechanism may not exist in hu-mans, because β-alanine did not inhibit human BUP.