The gene (termed daa) encoding N-acyl-D-aspartate (D-Asp) amidohydrolase (D-AAase) from the Alcaligenes xylosoxydans subsp. xylosoxydans (Alcaligenes A-6) was cloned in Escherichia coli (E. cell) JM109. The daa gene consists of 1,494 nucleotides and encodes 498 amino acid residues. The molecular weight of D-AAase was calculated to be 53,581. The N-terminal amino acid sequence (NH2-TDRSTLDDAP-) predicted by the nucleotide sequence matched exactly those of the purified D-AAase from both Alcaligenes A-6 and cloned E. coil, with the exception of the removal of the N-terminal methionine processed after translation. A comparison of the amino acid sequence of D-AAase with that of D-aminoacylase from Alcaligenes A-6 showed high overall homology (56%), D-AAase from Alcaligenes A-6 showed 25 similar to 29% homology with Bacillus stearothermophilus, porcine, and human L-aminoacylases. The daa was highly expressed in E. coli, and the recombinant enzyme was purified to homogeneity with 17.8% yield.